Altered Pattern of Proteolysis of Extracellular Proteins in Rhegmatogenous Retinal Detachment by Mining of Tryptic and N-Termini Datasets From Vitreous Humor Proteome

Altered Pattern of Proteolysis of Extracellular Proteins in Rhegmatogenous Retinal Detachment by Mining of Tryptic and N-Termini Datasets From Vitreous Humor Proteome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Altered Pattern of Proteolysis of Extracellular Proteins in Rhegmatogenous Retinal Detachment by Mining of Tryptic and N-Termini Datasets From Vitreous Humor Proteome Diego Sbardella, Gabriele Antonio Zingale, Sara Giammaria, Irene Pandino, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5395071/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Retinal detachment is a severe eye condition characterized by the detachment of the neurosensory retina from the retinal pigment epithelium and caused by retinal tears. Pars plana vitrectomy is the elective surgical procedure during which vitreous humor is collected. This fluid shapes the eye globe providing mechanical and nutritional support to the retina. Hence, exploring the proteome of vitreous humor isolated from subjects diagnosed with retinal detachment is supposed to help decipher the pathobiology of the disease and that of its complications, such as proliferative vitreo-retinopathy, which predispose to recurrent RD (observed in 20% of cases), a sight threatening condition. Herein, we investigated the perturbations of vitreous proteome between subjects affected by primary retinal detachment and controls by shot-gun proteomics approaches. Spectra were first searched and analyzed to identify proteome perturbations. Thereafter, starting from the hypothesis that the disease could be sustained by altered proteolytic processing of structural and non-structural elements of vitreous humor, N- and C-termini were mined to uncover endogenous proteolytic events. This search retrieved evidence of a wide repertoire of proteolytic events and proteolytic sites, either already described for proteins commonly identified also in other biological samples, or likely specific of this fluid. Comparison between the N- and C-termini landscapes and the perturbations of global proteome highlighted robust alterations of the repertoire of cleaved proteins between retinal detachment and control subjects. Strengthened by immunoblotting studies on a selection of proteins, datasets envisage that retinal detachment is characterized by unbalanced proteolysis of structural and non-structural components involved in the regulation of immune processes, proteolytic control and, in particular, angiogenesis. Biological sciences/Biochemistry Biological sciences/Biochemistry/Proteases Biological sciences/Biochemistry/Proteolysis Biological sciences/Biochemistry/Proteomics Health sciences/Biomarkers/Predictive markers Vitreous humor proteomics N-termini proteolysis angiogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Rhegmatogenous Retinal Detachment (RD) is a severe condition characterized by the detachment of the neurosensory retina from the Retinal Pigment Epithelium (RPE) due to the presence of retinal tears 1 – 4 . The incidence of RD is 7 to 12/10.000 cases per year 1 with a guarded prognosis and a heavy burden for the patients’ quality of life and health care expenditures 5 . Recurrent RD occurs in up to 20% of cases 1 , leading to severe visual loss and often requiring multiple surgery 1 . Proliferative Vitreo-Retinopathy (PVR) 1 is the leading cause of Recurrent RD (RRD) and of severe visual loss, and is characterized by the growth and contraction of cellular aggregates on the retinal surface and within the vitreous chamber 6 – 8 . The Epithelial to Mesenchymal Transformation (EMT) represents the hallmark of PVR and comprises the acquisition of morphological and functional properties of RPE cells, resulting in blood hemodynamics alterations, vessel microarchitecture changes, fibrosis and, eventually, retinal contraction 2 , 6 . The molecular footprint and the cascade of events promoting EMT and the pathological retinal remodeling associated to PVR has been extensively studied, yet is still far to be fully understood. Previous studies have shown changes in RPE and photoreceptors metabolism, cell adhesiveness and polarization, extracellular matrix turnover and remodeling, immune system cross-talk, redox unbalance, angiogenesis and inflammation 2 , 6 , 9 – 11 . Pars Plana Vitrectomy (PPV) is the elective surgical procedure for complex RDs and allows the collection of vitreous (VH) and aqueous (AH) humor samples. Research activities on these fluids are intrinsically complex since protein concentration is typically very low and identification of relevant proteins is hidden by abundant contaminating proteins (e.g., albumin, IgG etc.), often requiring sample pooling procedures and off-line separation or depletion of these proteins. Previous relevant proteomics studies applied to the human vitreous and specifically to RD patients suggested novel molecular perspectives on RD and PVR pathobiology 12 – 16 . However, to date, post-translational modification (PTM) profiling of VH samples has not yet (to the best of our knowledge) been performed. PTMs such as phosphorylation, glycosylation, and proteolytic processing are able to profoundly alter the structure and function of a protein 17 , 18 . In this regard, we reasoned that retinal detachment could be linked to the dysregulation of physiological PTMs and, in particular, endogenous proteolytic events carried out by the different classes of enzymes populating the VH. The present study reports the results of a pilot shotgun Label Free Quantification (LFQ) proteomic characterization of vitreous samples collected from primary RD patients (n = 8), compared to controls (n = 8) who underwent vitreoretinal surgery for formation of epiretinal membranes. Without immunodepleting for most abundant proteins and working on a single-subject scale, we identified a total 79 differentially expressed proteins (DEPs) between RD and Ctrl VH, uncovering precise alterations of key pathways for cell metabolism and tissue homeostasis. Thereafter, using proteomics technologies and analysis tools, 17 , 19 we explored the repertoire of endogenous N-termini (and C-termini). This approach identified several known and unknown sites of proteolysis in structural and non-structural VH components and significant alterations on proteins serving roles for immune system regulation, proteolytic balance and, in particular, angiogenesis. This scenario introduces the perspective that the pathogenesis of RD progresses through unbalanced extracellular proteolysis. METHODS Ethics Approval – Study Design The study was approved by the local ethics committee (P.R.O. Project, 09/14/2023). The main goal of the study is to collect vitreous and aqueous humour to investigate pathogenic determinants and potential biomarkers of most prevalent retinal disorders, including retinal detachment, by proteomics approaches. Samples collected are stored in an internal biobank of the institute. In accordance to the tenets of the Declaration of Helsinki, enrolled patients were asked to read and sign an informed consent form. Inclusion criteria for cases: age > 18 years, primary rhegmatogenous retinal detachment who underwent vitrectomy, phakic or pseudophakic patients. Inclusion criteria for controls: age > 18 years, patients who underwent vitrectomy for reasons different from retinal detachment (e.g., epiretinal membranes, dislocated intraocular lens), phakic or pseudophakic patients. Exclusion criteria (apply to both experimental groups): diabetes, collagenopathies (e.g. Ehlers-Danlos Syndrome), cancer, vitreous hemorrhages, concomitant neurodegenerative, inflammatory or infective eye diseases, alterations of the electrophoretic profile of the gamma-globulin band Vitreous samples were obtained from patients undergoing PPV for either retinal detachment or epiretinal membrane peeling. Approximately 1 mL of fluid were collected using a sterile 5 mL syringe and poured into a sterile 1.7 mL Eppendorf test tube. Samples were then immediately cleared by centrifugation and protein concentration quantified by bicinchoninic acid assay (BCA). Thereafter, samples were subdivided into different aliquots and stored at -80°C until use. No freeze/thaw cycles were applied for proteomics or Western blotting studies. All samples discussed in the study were processed and analysed in parallel. Anonymity of subjects was guaranteed throughout the entire study. Initially, n = 9 RRD cases and controls were enrolled in the study and analysed by mass spectrometry. One subject per group was then filtered out from analysis for technical issues during sample preparation. Raw mass spectrometry data of these samples were uploaded (corresponding to original samples #6 and #16) in the PRIDE repository (see “Data availability statement”) together with all samples effectively analysed and part of the study. Selection of samples was based on the best matching of demographic and clinical characteristics of the subjects. These data are introduced in the results section and further reported in Table I. Mass spectrometry In the case of proteomic analysis, samples (100 µg of VH proteins) were dehydrated in a SpeedVacuum system, reconstituted in denaturing buffer (6M guanidine-HCl, 50 mM Hepes, pH 7.8). Proteins were then reduced [5 mM dithiothreitol, 45 min, room temperature (r.t.)], alkylated (10 mM iodoacetamide, 30 min, r.t) and digested with trypsin (1:50 enzyme:protein ratio, overnight, 37°C) (Fisher Scientific, Waltham, MA, USA). Trypsin digestion was quenched with 0.4% trifluoroacetic acid (TFA) and peptides cleaned using Stage-Tips (C18 resin) (Fisher Scientific, Waltham, MA, USA). Thereafter, eluted peptides were dried by SpeedVacuum system and resuspended in 2% Acetonitrile, 0.05% Trifluoracetic Acid for mass spectrometry injection. Proteomic analysis was performed injecting (twice) 1 µg peptides for each experimental conditions into an Orbitrap Exploris 240 mass spectrometer coupled to an Ultimate 3000 nano-ultra high performance liquid chromatography (nano-UHPLC) system. Solvent A: 100% H 2 O, 0.1% Formic Acid; Solvent B: 80% Acetonitrile, 0.1% Formic Acid. UHPLC Gradient (minutes - %B): 0–6.7; 2–6.7; 62–34.4; 67–55.5; 72–100; 80–100; 82 − 6.7; 88 − 6.7. Column oven temperature: 45°C. Run time: 88 min. Loading Pump flowrate: 30 µL/min. NC Pump flowrate: 250 nL/min. Data acquisition was conducted in Data Dependent Acquisition (DDA) mode. Orbitrap Resolution: 120,000; Scan Range (m/z): 375–1650; RF Lens (%): 80; Normalized AGC Target (%): 300. ddMS² was triggered using the following filters: Isolation Window (m/z): 2; Normalized HCD Collision Energy (%): 30; Orbitrap Resolution: 15000; Normalized AGC Target (%): 50. Data and Statistical Analysis Protein and peptides were searched using Proteome Discoverer (PD) software (v. 2.5, Thermo Fisher Scientific) against a UniProt human protein FASTA database including protein isoforms. Sequest implemented with the Inferys rescoring algorithm was used and a concatenated target-decoy strategy applied for determination of the proteins False Discovery Rate (strict FDR ≤ 0.01 and relaxed FDR ≤ 0.05). Trypsin (full) was set as enzyme, 10 ppm precursor mass tolerance and 0.02 Da fragment mass tolerance. Carbamidomethylation of cysteines (+ 57.021) was set as static modification, whereas oxidation on methionine (+ 15.995) as dynamic modification per peptides according to the requirements of Inferys algorithm. The search of N-termini and C-termini was instead run using FragPipe/MsFragger (v. 22.0), as reported elsewhere 18 . The resulting protein and peptides identifications and quantitation data were analysed using an in-house built R script (v. 2.5). In-depth statistical analysis for differential protein expression between RD and control groups are described throughout the results section. Western blotting studies A selection of proteins identified by the proteomic approach was further analyzed by denaturing and reducing Western blotting using an aliquot of VH not denatured by guanidine-HCl as described before. In all cases, 5 µg of vitreous proteins per subject were heat-denatured and reduced in Laemmli buffer 1x supplemented with 1 mM dithiothreitol (DTT). Thereafter, 4–20% acrylamide pre-cast gels (Bio-Rad, Hercules, CA, USA) were used to separate proteins by SDS-PAGE. After separation, proteins were transferred to a HyBond-ECL nitrocellulose filters (Bio-Rad, Hercules, CA, USA) and probed with the antibodies indicated. All antibodies used were purchased from ProteinTech (Rosemont, Illinois, USA). Antibodies were diluted 1:3000 in 0.1% Tween-PBS 0.1% fat-free milk and with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody (Bio-Rad, Hercules, CA, USA), diluted 1:10.000 in 0.1% Tween-PBS 0.1% fat-free milk. Proteins were developed by ECL chemiluminescence and recorded in a iBright 1500 (ThermoFisher scientific). RESULTS Subjects enrollment and study design Vitreous samples were obtained from patients undergoing PPV for either RD (RD, n = 8, mean age 69 ± 4 years) or macular pucker (n = 8, mean age 75 ± 6 years), enrolled as controls (Ctrl). Additional demographic, epidemiological and clinical data of enrolled patients are summarized in Table 1 . Systemic hypertension was the prevalent co-morbidity across enrolled subjects. A heterogeneous localization (supero-temporal and inferior) of the retinal detachment was documented in the RD group. Table 1 Demographic and clinical parameters of subjects’ enrolled in the study. ID Gender Age* Comorbidity Systemic Therapy VR Disease Eye Visual Acuity** Pseudophakia RD #1 M 68 Systemic Hypertension, Gilbert syndrome β-blocker, Ca 2+ channel blocker, ARB Supero-Temporal RD, Vitreal Proliferation Signs, Macula Off R Motu Manu No RD #2 M 74 / / Infero-Temporal RD, Vitreal Proliferation Signs, Macula Off R Motu Manu Yes RD #3 M 73 Systemic Hypertension, myocardial infarction Sartan, β-blocker, Xa Factor inhibitor, Antiarrhythmic Supero-Temporal RD, Vitreal Proliferation Signs, Macula Off R Light Perception No RD #4 M 67 Systemic Hypertension β-blocker Supero-Temporal RD, Vitreal Proliferation Signs, Macula Off L Light Perception No RD #5 F 73 Systemic Hypertension, Osteoporosis Hydrochlorothiazide + sartan, Vitamin D Subtotal RD, Macula Off R 1/20 Yes RD #6 F 62 Systemic Hypertension, myocardial infarction, deep vein thrombosis β-blocker, Xa Factor inhibitor Infero-Temporal RD, Macula On R 3/10 No RD #7 F 67 / / Inferior RD, Macula Off L Motu Manu Yes RD #8 M 65 Systemic Hypertension β-blocker, ACE Inhibitor Inferior RD, Vitreal Proliferation Signs, Macula Off R Motu Manu Yes CTRL #1 M 80 Systemic Hypertension, Dysthyroidism, Hypercholesterolemia, Benign prostatic hypertrophy β-blocker, levothyroxine, 5 alpha reductase inhibitor, Statin Epiretinal Membrane, Cataract R 5/10 Yes CTRL #2 M 81 / / Epiretinal Membrane, Cataract L 3/10 No CTRL #3 F 82 / / Epiretinal Membrane, Cataract L 4/10 No CTRL #4 F 78 Systemic Hypertension, Atrial fibrillation Proton pump inhibitor, β-blocker, ACE inhibitor Dislocated IOL L 1/20 No CTRL #5 M 72 / / Epiretinal Membrane, Cataract L 1/10 No CTRL #6 F 72 Systemic Hypertension, Hypercholesterolemia, Osteoporosis Hydrochlorothiazide + Sartan,Statin, Vitamin D Macular Hole, Epiretinal Membrane, Cataract R 1/10 No CTRL #7 M 68 / / Macular Hole, Epiretinal Membrane, Cataract L 1/10 No CTRL #8 M 68 / / Epiretinal Membrane, Cataract L 1/10 No *Years RD = Retinal Detachment **Decimals IOL = Intraocular lens An equal quantity (µg) of VH protein for each enrolled sample was digested with trypsin and subjected to proteomics analysis. A workflow of the study is summarized in Fig. 1 . Dysregulation of metabolic pathways and structural components of the ECM in VH from RD patients Identification by MS yielded a total 2,134 proteins with ≥ 1 unique peptides together with 7,162 peptide groups and 287,667 PSMs. Results were then filtered for Master proteins, as they are defined in PD glossary. In total 798 proteins were identified, with a robust overlap between RD and Ctrl: 745 proteins were common to the two experimental groups, 22 proteins were documented as exclusive of RD group and 31 of the Ctrl group (discussion of exclusive proteins is limited to those identified in ≥ 50% subjects/group) (Fig. 2 A). In this regard, supplementary Tables 1 and 2 report the list of proteins exclusive for RD and Ctrl groups, respectively. The distribution of protein intensities was verified upon log 2 transformation (Fig. 2 B). Density-plots of non-normalized intensities, though highlighting moderate intragroup variability, suggested a comparable intra-group and inter-group normal distribution for both RD and Ctrl datasets. Therefore a class-specific quantile normalization strategy was deemed applicable 20 . The experimental variability improved, as shown in the post-quantile normalization density plot (Fig. 2 C). Thereafter, missing values were imputed using Classification and regression trees (CART) approach 21 . Data were then analyzed by Principal Component Analysis (PCA) to further check for global differences between RD and Ctrl experimental groups; data distribution and the position of the group-specific centroid suggested that the biological data had indeed different features (Fig. 2 D). DEPs between RD and Ctrl subjects were then analyzed using a moderate Bayesian t test LIMMA statistical approach and setting as threshold for significance log 2 FC ≤ 0.57 and p ≤ 0.05 adjusted by Benjamini-Hochberg correction (Fig. 3 ). In total, 43 proteins were found as upregulated and 36 as downregulated in the RD group, compared to Ctrl (DR/Ctrl ratio); the entire list of upregulated and downregulated proteins in RD and Ctrl VH is shown in Tables 2 and 3 , respectively. To better cluster and rationalize data, DEPs discussed above and group-exclusive proteins identified and quantified were submitted to gene ontology (GO) to infer statistically enriched (p ≤ 0.05) molecular function (MF) (Fig. 4 A,B), cellular components (CC) (Suppl. Figure 1), and biological processes (BP) (Suppl. Figure 2). Table 2 Proteins upregulated in RD VH. The table shows the Accession number (UniProt), the log2FC calculated for the RD/Ctrl ratio, the p.ordinary (p.ord) value calculated by applying the LIMMA test, the Protein Description, the number of peptides identified and the calculated FDR. Accession logFC p.ord Description # Peptides FDR P10745 1.639 0.03291 Retinol-binding protein 3 63 ≤ 0.01 E7EUF1 1.096 0.04215 Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 34 ≤ 0.01 P01011 1.320 0.01991 Alpha-1-antichymotrypsin 31 ≤ 0.01 A0A1B0GWE8 1.508 0.02229 Cathepsin D 29 ≤ 0.01 P10523 4.124 0.00014 S-arrestin 27 ≤ 0.01 P36222 2.473 0.01013 Chitinase-3-like protein 1 26 ≤ 0.01 B4DPQ0 1.215 0.03967 Complement subcomponent C1r 25 ≤ 0.01 P02788 2.444 0.01314 Lactotransferrin 22 ≤ 0.01 Q17R60 3.445 0.00001 Interphotoreceptor matrix proteoglycan 1 22 ≤ 0.01 P02766 0.727 0.01162 Transthyretin 20 ≤ 0.01 A0A7I2V2D2 1.342 0.04520 Plasma protease C1 inhibitor 20 ≤ 0.01 P62873 3.057 0.01086 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 19 ≤ 0.01 P02751 1.082 0.02407 Fibronectin 18 ≤ 0.01 P01834 0.702 0.04086 Immunoglobulin kappa constant 16 ≤ 0.01 P08571 1.363 0.00640 Monocyte differentiation antigen CD14 15 ≤ 0.01 Q15582 1.996 0.04921 Transforming growth factor-beta-induced protein ig-h3 13 ≤ 0.01 P10451 1.133 0.04296 Osteopontin 12 ≤ 0.01 P07858 1.357 0.02937 Cathepsin B 12 ≤ 0.01 Q9BZV3 3.318 0.00486 Interphotoreceptor matrix proteoglycan 2 12 ≤ 0.01 P69905 3.387 0.04104 Hemoglobin subunit alpha 12 ≤ 0.01 P01033 1.669 0.01652 Metalloproteinase inhibitor 1 11 ≤ 0.01 Q6EMK4 2.303 0.00156 Vasorin 11 ≤ 0.01 Q92743 1.394 0.04816 Serine protease HTRA1 10 ≤ 0.01 P07602 1.801 0.01956 Prosaposin 10 ≤ 0.01 P61626 0.947 0.01846 Lysozyme C 9 ≤ 0.01 P04040 1.480 0.04360 Catalase 8 ≤ 0.01 P05546 1.184 0.02050 Heparin cofactor 2 6 ≤ 0.01 Q96JP9 2.234 0.00677 Cadherin-related family member 1 6 ≤ 0.01 P55058 1.941 0.00004 Phospholipid transfer protein 5 ≤ 0.01 P01619 0.647 0.03826 Immunoglobulin kappa variable 3–20 4 ≤ 0.01 Q9HCQ7 2.576 0.00492 Pro-FMRFamide-related neuropeptide VF 4 ≤ 0.01 Q8N114 3.473 0.00066 Protein shisa-5 4 ≤ 0.01 H0YCV9 0.792 0.04504 CD44 antigen (Fragment) 3 ≤ 0.01 Q9BYJ0 1.665 0.02877 Fibroblast growth factor-binding protein 2 3 ≤ 0.01 P02747 1.094 0.03152 Complement C1q subcomponent subunit C 2 ≤ 0.01 A0A0C4DH73 1.171 0.02652 Immunoglobulin kappa variable 1–12 2 ≤ 0.01 P17936 1.213 0.01349 Insulin-like growth factor-binding protein 3 2 ≤ 0.01 Q9NP84 1.582 0.03531 Tumor necrosis factor receptor superfamily member 12A 2 ≤ 0.01 P84243 1.906 0.01618 Histone H3.3 2 ≤ 0.01 Q8NA57 1.156 0.03437 Uncharacterized protein C12orf50 1 ≤ 0.05 P81172 1.653 0.03782 Hepcidin 1 ≤ 0.01 J3KQ66 3.261 0.02705 Reelin 1 ≤ 0.01 P10586 3.624 0.02274 Receptor-type tyrosine-protein phosphatase F 1 ≤ 0.05 Table 3 Proteins downregulated in RD VH. The table shows the Accession number (UniProt), the log 2 FC calculated for the RD/Ctrl ratio, the p.ord value calculated by LIMMA test, the Protein Description, the number of peptides identified and the calculated FDR. Accession logFC p.ord Description # Peptides FDR P53674 -3.506 0.02262 Beta-crystallin B1 42 ≤ 0.01 P00352 -3.406 0.01493 Aldehyde dehydrogenase 1A1 42 ≤ 0.01 P43320 -2.842 0.04370 Beta-crystallin B2 37 ≤ 0.01 P48637 -3.246 0.00414 Glutathione synthetase 31 ≤ 0.01 P22914 -2.703 0.04731 Gamma-crystallin S 30 ≤ 0.01 P05813 -3.910 0.03066 Beta-crystallin A3 27 ≤ 0.01 P29401 -3.471 0.01115 Transketolase 25 ≤ 0.01 P26998 -2.312 0.02451 Beta-crystallin B3 24 ≤ 0.01 P53673 -2.784 0.02749 Beta-crystallin A4 20 ≤ 0.01 Q00796 -2.797 0.04260 Sorbitol dehydrogenase 18 ≤ 0.01 P53672 -4.941 0.00722 Beta-crystallin A2 17 ≤ 0.01 P16152 -4.295 0.03914 Carbonyl reductase [NADPH] 1 17 ≤ 0.01 Q01082 -2.667 0.03033 Spectrin beta chain, non-erythrocytic 1 16 ≤ 0.01 P04792 -3.959 0.00299 Heat shock protein beta-1 11 ≤ 0.01 Q71U36 -2.350 0.04348 Tubulin alpha-1A chain 11 ≤ 0.01 P09936 -4.167 0.01810 Ubiquitin carboxyl-terminal hydrolase isozyme L1 8 ≤ 0.01 Q93088 -3.577 0.00427 Betaine–homocysteine S-methyltransferase 1 8 ≤ 0.01 Q96GW7 -2.696 0.00185 Brevican core protein 8 ≤ 0.01 P15121 -3.489 0.00197 Aldo-keto reductase family 1 member B1 7 ≤ 0.01 P07900 -2.368 0.02553 Heat shock protein HSP 90-alpha 6 ≤ 0.01 P22061 -3.692 0.02398 Protein-L-isoaspartate(D-aspartate) O-methyltransferase 5 ≤ 0.01 P14174 -2.567 0.00727 Macrophage migration inhibitory factor 5 ≤ 0.01 P22392 -2.535 0.03512 Nucleoside diphosphate kinase B 5 ≤ 0.01 P63104 -2.511 0.02565 14-3-3 protein zeta/delta 5 ≤ 0.01 A0A8Q3WKK9 -2.820 0.02229 Lactase-like protein 3 ≤ 0.01 Q9NS15 -2.782 0.02936 Latent-transforming growth factor beta-binding protein 3 3 ≤ 0.01 Q16555 -2.719 0.00993 Dihydropyrimidinase-related protein 2 3 ≤ 0.01 Q14019 -2.415 0.01600 Coactosin-like protein 3 ≤ 0.01 B5MDF5 -2.498 0.04477 GTP-binding nuclear protein Ran 2 ≤ 0.01 Q05639 -2.494 0.03265 Elongation factor 1-alpha 2 2 ≤ 0.01 E9PQW4 -2.265 0.01110 Mitogen-activated protein kinase 2 ≤ 0.01 D6RA82 -5.687 0.00041 Annexin 1 ≤ 0.05 H7C4I3 -5.215 0.00003 RuvB-like helicase (Fragment) 1 ≤ 0.05 Q86SR1 -4.559 0.03738 Polypeptide N-acetylgalactosaminyltransferase 10 1 ≤ 0.05 P37837 -2.236 0.04249 Transaldolase 1 ≤ 0.01 P03973 -2.121 0.03092 Antileukoproteinase 1 ≤ 0.01 With regard to proteins upregulated in RD VH, it is worth mentioning extracellular matrix components such as interphotoreceptor matrix proteoglycan 1 and 2 (Q17R60 and Q9BZV3, respectively) and fibronectin (P02751). Moreover, proteins involved in TGF-β signaling and pro-inflammatory cascades or angiogenic processes such as transforming growth factor-beta-induced protein Ig-h (Q15582), fibroblast growth factor binding protein (Q9BYJ0), vasorin (Q6EMK4) and osteopontin (P10451) were found upregulate together with complement factors and natural inhibitors of peptidase and protease activity, such as metalloproteinase inhibitor 1 (P01033), α1-antichymotrypsin (P01011) and heparin cofactor 2 (P05546). Accordingly, GO term analysis identified enrichment (p ≤ 0.05) of the following terms: collagen-containing extracellular matrix (GO:006023), photoreceptor outer segment and cell cilium (GO:0001750 and GO:0097733, respectively), laminin (GO:0043236) and fibronectin binding (GO:0001968), enzyme inhibitor activity (GO:0004857) peptidase inhibitor and endopeptidase regulatory activity (GO:0030414 and GO:0061135, respectively). Conversely, proteins downregulated in RD VH were mostly involved in proteostasis regulation and energetic/metabolic cycles, including carbonyl reductase (P16152), transketolase and transaldolase (P29401 and P37837, respectively), glutathione synthetase and reductase (P48637 and P00390, respectively), stress proteins, such as heat shock protein 90 (P07900), β-crystallins A3 and B1 (P05813, P53674, respectively) and proteins involved in ubiquitin (Ub) signaling, such as lengsin (Q5TDP6), ubiquitin carboxyl-terminal hydrolase isozyme L1 (P09936). Accordingly, GO charts highlighted an enrichment in terms referable to mechanisms of proteostasis regulation and energetic metabolism such as NAD binding, (GO:0051287), protein folding chaperone (GO:0044183), oxidoreductase activities (GO:0016616, GO:0016614), Mining of N-termini highlights dysregulation of proteolysis in vitreous humor from RD patients Given the enrichment of peptidases and inhibitors that was observed in functional enrichment analysis of DEPs, we hypothesized that RD patients may suffer from unbalanced extracellular proteolysis. To test this hypothesis, FragPipe/MSFragger was used to search for semi-tryptic peptides indicative of proteolytic processing, with the specific aim of identifying N-termini and C-termini. A peptide-level FDR ≤ 0.01 was applied, and the main criteria for N-termini identification were set for the absence of a lysine (K) or arginine (R) residue at the N-terminal flanking position of the peptide. This last search parameters were set to filter out peptides probably generated by the trypsin digestion of samples, thus not representing bona fide endogenous N-termini. The search parameters chosen for the analysis enabled the identification of two classes of termini: mature N-termini, which, in most cases, originate from the enzymatic processing (often intracellular) of the signal peptide during protein secretion, and neo N-termini, which typically correspond to enzymatic cleavage of mature proteins. Upon further filtering for N-termini identified in at least 3 out of 8 subjects/group, the search identified a total of 457 peptides common to RD and Ctrl VH, 182 exclusive of RD and 216 of Ctrl VH samples (Fig. 5 A). Neo N-termini were more represented than mature N-termini in both experimental groups. N-termini documented as exclusive of either one of the two groups, and N-termini upregulated or downregulated (LIMMA test, Supplementary Fig. 3) were submitted (separately for each group) to TopFINDER (v. 4.1, https://topfind.clip.msl.ubc.ca/ ) to search for known cleavage sites of proteins identified and putative enrichment of specific proteases (the complete name of enzyme discussed below is summarized in Table 4 ). Table 4 Lists of enzymes and their abbreviations Acronym Enzyme Name MMP2, MMP9 Matrix Metalloproteinase 2, 9 (Gelatinases) MMP3, MMP7 Matrix Metalloproteinase 3, 7 MMP12 Matrix Metalloproteinase 12 BMP1 Bone Morphogenic Protein 1 CATH-D, -S, -L, -B Cathepsin-D, -S, -L, -B KLK Kallikrein MEP1B Meprin 1B ADAMTS-10, -17 A Disintegrin and Metalloproteinase with Thrombospondin motifs-10, -17 BACE Beta-secretase 1 A separated discussion for N-termini enriched/exclusive of either Ctrl or RD subjects is then provided for the sake of the readership. Several previously characterized neo N-termini were identified as downregulated (-0.57 Log 2 FC, p adj ≤0.05) in the RD/Ctrl ratio or specific (assigned for those identified and quantified in ≥ 75% subjects) of Ctrl VH samples (Supplementary Table 3). They include: i) several (n = 15) previously characterized neo-terminal fragments of crystallin αB released by MMP9; ii) a wide array of proteolytic fragments of additional crystallins, such as γ-crystallins (isoforms 2 − 1, 4 and S, P07315, P07320 and P22914, respectively), β-crystallin (P43320) and heat shock protein β4 (P02489); iii) a neo N-terminus of serpin-B9; iv) a previously unknown neo N-terminus of protein bassoon (Q9UPA5), which is a presynaptic protein and major component of photoreceptor ribbon. In this case, the potential cleavage site was mapped between 1228 ↓Tyr 1229 ; v) a neo N-terminus of osteopontin (P10451-4). This fragment is generated by cleavage of the protein at 185 ↓Ala 186 by MMP3/MMP7 22 . Interestingly, although the observation did not reach statistical significance, a previously unknown neo N-terminus of interphotoreceptor matrix proteoglycan 2 (IMPG2) (Q9BZV3) was found enriched in Ctrl VH (Supplementary Table 4). With regard to mature N-termini enriched in Ctrl VH, it is worth documenting the identification of Wnt1 inhibitory factor (Q9Y5W5), secreted frizzled-related protein 3 (DKK3) (Q92765), spondin-1 (Q9HCB6), oligodendrocyte myelin glycoprotein (Q5SSB8), APP amyloid-beta precursor protein (P05067) and isoform 2 of fibrinogen alpha chain (A0A0S2Z3E8). In most cases, these findings were consistent with the tryptic dataset (Supplementary Table 3). In the case of VH samples of RD subjects, although a very limited number of upregulated N-termini (DR/Ctrl ratio, ≥ 0.57 Log 2 FC, padj ≤ 0.05) was identified, mature N-termini of several enzyme inhibitors and immune system components were documented (Supplementary Table 5): i) C3 and PZP-like alpha-2-macroglobulin domain-containing protein 5 (P01023); ii) α-1-antichymotrypsin (P0101); iii) complement factor B (P00751); iv) cystatin-3 (P01034); v) CSF1 receptor (P07333); vi) retinol binding protein (P10745); vii) insulin binding proteins-4 (P22692); vi) serum amyloid A protein (P0DJI8). In this last case, the peptide was previously reported to be released by the CATL1, MMP3 and CATB cut at residues 22 ↓Ser 23 . Looking beyond the neo-termini which were specific of RD VH group (focusing on those identified and quantified in ≥ 50% subjects/group) highlighted several known and unknown peptides serving roles for the regulation of cell adhesiveness and, most notably, angiogenesis (Supplementary Table 5), yielding insights into the proteolytic landscape of vitreous humor: i) several proteolytic fragments of plasma proteins such as Albumin (P02768), including a previously unknown fragment generated by MEP1B and BACE2 cut between residues 174 ↓Ala 175 ; ii) serotransferrin (P02787) identified by two legumain cleavage sites mapped between residue 601 ↓Asn 602 and 602 ↓Pro 603 ; iii) a fragment of complement C1r subcomponent (P00736), with a cleavage site located between residues 186 ↓His 187 ; iv) a fragment of clusterin (isoform 2, P10909-2) for which a cleavage fragment generated by MMP-3 and MMP12 at residues 309 ↓Met 310 was documented; v) calsyntenin-3 (isoform 2, Q9BQT9-2) reporting a cleavage site of ADAMTS-10 and − 17 at residues 824 ↓Val 825 ; vi) apolipoprotein A1 showing a MMP7 and MMP12 cleavage site at residues 224 ↓Ser 225 ; vii) fibronectin A - isoform2 for which two cleavage sites of MMP2, MMP8, MMP13 at residues 19 ↓Ala 20 and 20 ↓Asp 21 were documented; viii) basement membrane-specific heparan sulfate proteoglycan core protein (P98160). In this case, a peptide spanning across residue 4196–4221 within the C-terminal portion of the molecule and a potential cleavage site between residues 4195 ↓Glu 4196 was identified. The fragment, called endorepellin, has been previously characterized to be released by BMP1 and to possess potent antiangiogenic activities 23 ; ix) vitronectin (P04004), with a potential cleavage site not previously reported and located between residues 422 ↓Asp 423 and falling within the cell adhesion domain (hemopexin domain 4). To circumstantiate further these findings, cleavage sites derived from protein termini of RD and Ctrl groups were analyzed by a sequence logo plot (Fig. 5 B,C). A net prevalence of polar residues, in particular glycine (G) and serine (S) was observed in P1 and P1’ position in the case of Ctrl VH (Fig. 5 B). Conversely, RD VH samples show a robust prevalence of polar residues such as alanine (A) and valine (V) over the same positions (Fig. 5 C). These preferences were confirmed also proceeding towards the P5 position, suggesting that different proteases may contribute to the proteolytic events of VH between RD and Ctrl. Mining of proteolytic events was then implemented with the search of C-termini by applying again a peptide-level FDR ≤ 0.01. In this case, the main criteria for identification were the absence of a K or R residue at the C-terminus of the peptide. Mining of C-termini further expanded coverage of the VH degradome. In total, 149 C-termini were found as common to both the experimental groups, 46 exclusive of RD and 130 exclusive of Ctrl (Fig. 5 D). Whilst no significantly upregulated or downregulated C-termini were identified, in the case of Ctrl VH, C-termini for several crystallin proteins were detected, including a previously characterized MMP9 cleavage site within crystallin αB which is complementary to one of the neo N-termini identified for the same protein and discussed above. The full list of C-termini identified is provided in Supplementary Table 6. Figure 6 shows a schematic representation of biologically relevant N-termini and C-termini cleavages sites within the domain of proteins identified. Western blot validation of protein identification and proteolytic processing of osteopontin and vitronectin Proteolytic processing of proteins was validated with manageable electrophoretic features (for this reason perlecan was excluded from analysis) (Fig. 7 ). For each sample, 5 µg of VH proteins were separated and probed with antibodies raised against osteopontin (OPN), vitronectin (VTN), interphotoreceptor matrix proteoglycan 1 and 2 (IMPG-1 and IMPG-2, respectively) and DKK3. Given the semi-quantitative nature of the methodological approach and, at least in our opinion, the lack of a valid internal control for normalization purposes, we prefer not to speculate on quantitative data between groups. In the case of osteopontin, the immunostaining highlighted the typical pattern of the full-length protein (i.e., > 2 bands). Although an apparent increase of full-length osteopontin intensity in RD VH was observed, a long-exposure of filters highlighted the presence of a faint ~ 30 kDa band only in the Ctrl group. This species was previously reported to correspond to the fragment released by MMPs degradation of the full-length protein 22 , which also corresponded in MW to the cleavage site identified by mining of N-termini. Thus, Western blot analysis validated our proteomic and N-termini mining workflow. In the case of vitronectin, the full-length protein again showed a slight apparent increase in the RD VH group. However, in this case, several putative fragments were immunostained by the antibody over lower molecular weight regions of the filter exclusively in the RD group as well. In the case of IMPG1 and IMPG2 the pattern observed was unequivocal. The two full-length proteoglycans were in facts detected exclusively in RD samples. However, in the case of IMPG1 an additional band of approximately 50 kDa was identified across all samples. In the case of IMPG2 several immunoreactive bands were documented at MW lower than that of the full-fragments generated by cleavage of the glycoproteins within the sperm protein, enterokinase and agrin (SEA) domain could not be immunostained nor in Ctrl or RD samples, a putative fragment (< 45 kDa) with intensity comparable across all samples was documented for IMPG1 and diffuse fragments with unclear identity were stained for IMPG2. With regard to DKK3, whilst the band intensity showed comparable between samples, different potential cleavage patterns were observed especially for Ctrl VH samples. DISCUSSION In this proteomic study, we coupled the characterization of VH proteome by LFQ DDA approaches with mining of natural N-termini (and C-termini), to introduce novel perspectives on the pathobiology of RD based on the identification of global perturbations of the proteome and of endogenous proteolytic events. Our global analysis of the VH proteome was performed on a single subject scale (rather than pooled samples) and without immunodepleting for the most contaminating proteins. Despite the technical challenges associated with analysis of lower protein quantities of higher dynamic range, we were able to identify a robust number of proteins that was consistent with the existing literature, confirming key findings and further introduced novel observations 12 , 13 , 24 . Previous studies on RD and, in particular, PVR, agreed on a multifaceted dysregulation of energetic metabolism, redox unbalance, immune system regulation and growth factors bioavailability 12 , 13 , 24 . Our study identified numerous dysregulated proteins and functionally enriched terms, with a sharp distinction between RD and Ctrl VH proteome. Several proteins involved in TGF signaling, which is the main pro-fibrotic cytokine, or in pro-inflammatory cascades, such as TNF receptors, were also identified as markedly enriched in the RD VH proteome. Interestingly, the polarization of macrophages towards the M2 phenotype has already been proposed as a driver of fibrosis and a major stimulus of RPE EMT during PVR; molecular cascades were effectively linked to TGFβ signaling 11 . A deeper examination of terms of CC, MF and BP processes, generated by submitting proteins upregulated and exclusive in RD VH to GO, highlighted further mechanisms that are very likely to underscore RD pathogenesis: A wide repertoire of terms describing mechanisms of turnover and remodeling of the extracellular matrix, with particular emphasis on the photoreceptor outer segment (interphotoreceptor matrix proteoglycans), structural proteins (collagen, fibronectin) and receptors involved in cell adhesiveness and motility (e.g., selectins) were documented significantly enriched. In addition, mechanisms or regulation of peptidase and protease activity were observed, this being in accordance with previous findings, indeed 15 , and posing a rationale for subsequent exploration of endogenous proteolytic events. In addition to yielding insights into the global VH proteome, our dataset also allowed us to mine evidence of post-translational modification of VH proteins by proteolytic processing. Natural endogenous N- and C-termini represent a minor fraction (~ 5%) of identifiable peptides within a proteome. Prior to the relatively recent release of ultrafast search algorithms such as MSFragger, a good coverage of these peptides was only achieved by adopting enrichment strategies, such as N-tails 17 . Although this enrichment procedure would almost certainly boost the numbers of identified N-termini, the informatic pipeline adopted by us is recognized to retrieve a robust coverage of the N-terminome 18 . Effectively, we here identified and quantified > 500 potential N-termini and > 300 C-termini. In most cases, they represent neo N-termini of structural and non-structural components of the VH that have not been reported before likely because the endogenous proteolysis of this fluid is an unexplored topic. However, several known cleavage sites were also identified. All together these data point out to specific events which may characterize the patho-physiological remodeling of the vitreous fluid. To test for specific enrichment of one or more proteases, data were submitted to TopFINDER (v. 4.1 https://topfind.clip.msl.ubc.ca/ ) 25 , a software specifically developed to characterize proteolytic events. This search retrieved evidence of the proteolytic activities of MMP9, CATD, CATS, MMP3 and MMP7 in Ctrl VH. A much broader array of enzymatic activities, including MMP12, MMP3, legumain, MEP1B, BMP1, were documented in RD VH. Although none of these proteases reached the statistical threshold (q ≤ 0.05) for being considered dominant, the possibility of unbalanced proteolysis in RD VH, in our opinion, is robustly supported by four unrelated findings, namely: i) abundant VH proteins, such as crystallins, albumin and serotransferrin show different trends. Whilst neo N-termini of crystallins were significantly enriched in Ctrl VH, this being consistent with the tryptic datasets, neo N-termini of albumin and serotransferrin were more represented in RD VH in the absence of any obvious protein accumulation, at least based on inspection of the tryptic dataset; ii) the distribution of cleavage preferences between RD and Ctrl samples. Hydrophobic residues ranked first across P5 to P1’ position of neo N-termini of Ctrl VH, whereas polar residues were mostly represented over the same sequence in the case of RD VH neo N-termini; iii) several mature and neo N-termini of natural inhibitors of proteases, such as α1-protease inhibitor, α1-antichymotrypsin, were identified in RD VH; iv) the pattern of neo N-termini of structural and non-structural elements was fairly different between the two experimental groups. With respect to this last point, the total repertoire of N-termini (including RD and Ctrl) suggests that proteolysis may in particular affect mechanisms of immune system regulation and cell adhesiveness/angiogenesis. Regarding the immune system, it is worth pointing out that mature and neo-termini, either C- or N-, were identified for complement components together with a previously identified neo N-terminus of natural killer cell-enhancing factor B in RD VH samples. Considering also the findings retrieved by the tryptic searches discussed above, it is likely that dysregulation of immune system polarization progressively develops in RD subjects. Discussion of proteolysis-mediated mechanisms of angiogenesis regulation, instead, is enriched with several interesting findings. A neo N-termini stretching across the 186–193 residues of osteopontin (assigned to isoform 4 by FragPipe but shared with wild type osteopotin) and previously described to be released by a MMP3/MMP7 cut at 185 ↓Ala 186 was identified in Ctrl VH. The cleavage of osteopontin was reinforced by the identification of a further C-terminus of the protein (in both Ctrl and RD VH) and by the Wb approach, which highlighted the presence of a faint 30 kDa fragment, but compatible with that released by MMP3 and MMP7 22 . Osteopontin is a sialoprotein serving key roles for the physiological composition of extracellular matrix 26 . Importantly, the fragment here detected was reported to have bioactive properties for cell adhesiveness, migration and angiogenesis. Remarkably, based on search on tryptic peptides, osteopontin was documented as robustly upregulated in the RD VH, suggesting that these subjects may experience an increase in protein level through reduced proteolysis. In a previous study, protein levels were strongly increased in the VH of PVR subjects, compared to subjects without this complication. This finding stimulated the authors to associate osteopontin levels to RD, probably through impaired angiogenesis 10 , 27 . In coherence with this scenario, two major structural glycoproteins, such as basement membrane-specific heparan sulfate proteoglycan core protein, also called perlecan, and vitronectin were identified with neo N-termini in RD VH. Perlecan, is a major component of the inner limiting membrane (ILM) and VH body. Correct synthesis and deposition of this glycoprotein, as well as its turnover, are key for the homeostasis of these two ECM components of the eye 28 , 29 . In our study, a perlecan neo N-terminus (4196–4222 residues) was found to stretch across the C-terminal fragment of the protein. This fragment, called endorepellin, is released by BMP1 cleavage at 4195 ↓Glu 4196 and was reported to have strong angiostatic activities by inhibiting endothelial cell adhesion to fibronectin and type I collagen 23 , 30 . Vitronectin, instead, is a glycoprotein synthesized and released by photoreceptors, but also a component of plasma 31 which play pivotal roles in ECM stabilization as well as regulation of vessels genesis and sprouting across the matrix layers. Vitronectin fragments were identified as neo N- and C-termini in RD VH and a possible fragmentation pattern of the protein was observed also by Wb, although, the abundance of the full length protein, by this approach, was probably higher in RD vs Ctrl VH. Nevertheless, dysregulated vitronectin levels were documented by a proteomic characterization of the VH isolated from patients with blood veins occlusions, confirming that even in the retina, the glycoprotein is expected to serve roles in vessels microarchitecture and, thereby blood hemodynamics. As widely discussed throughout the manuscript, these are both patho-physiological processes considered altered in RD and PVR subjects 32 – 34 . In addition to these main findings, a proteolysis-based dysregulation of angiogenesis is supported by additional evidence, including detection of neo N-terminus of cystatin 3, which promotes angiogenesis, and of PEDF (serpin F1), in RD VH. Among N-termini identified, it is worth commenting the case of IMPG1 and IMPG2. These are (both) secreted and membrane glycoproteins that serve key role for the physiological composition and homeostasis of the interphotoreceptor matrix which surround the inner and outer segment of photoreceptors. Interestingly, the biological activity of these glycoproteins is dependent on the proteolytic activation of the immature protein by an enzymatic cut within the SEA domain. Mutations in the SEA domain that impair proteolytic processing of IMPG1 and IMPG2 are associated with retinitis pigmentosa (RP) 35 . In this study, IMPG1 and IMPG2 were documented as robustly upregulated (Log 2 FC > 3, p.ord ≤ 0.05) in RD VH, compared to Ctrl VH, when the search was run using the tryptic dataset, confirming previous studies 13 , 16 , and exclusive of RD VH, at least for the sensitivity of the approach, by Wb. By deeply examining the PSMs, it is remarkable that, in the case of IMPG2, 12 high confident (FDR ≤ 0.01) tryptic peptides were observed in RD VH, and only 3 in Ctrl VH. However, in the case of IMPG2, a neo N-terminus was instead identified as robustly downregulated in RD VH (Log 2 FC = -0.734, p.ord < 0.444), whilst a neo C-terminus of the same protein was exclusively identified in Ctrl VH. The discrepancy between the tryptic and neo termini datasets envisages the possibility that accumulation of IMPG2 in RD VH, which is likely caused by photoreceptor degeneration, is not accompanied by their proteolytic processing, which is, instead, key for their biological function. This study has some limitations to acknowledge. Pitfalls of present study include the relatively small sample size and the absence of longitudinal data limiting commentary on their link to disease progression, in particular PVR. Larger well-controlled studies are required to confirm these pilot findings. An additional hypothesis that demands careful attention for the interpretation of this set of data and the additional ones that will be generated by forthcoming studies is that some of the proteins observed may display divergent compartmentalization within the same fluid. This is the case of extracellular vesicles (e.g., exosomes), which are released as a consequence of the degeneration the photoreceptors partially undergo during the acute phase of RD 36 . Sequestration of a protein inside a vesicle shields it from digestion and identification. Furthermore, many of the proteins here identified are heavily glycosylated under physiological conditions. Glycosylation is a PTM which critically protects them from proteolytic digestion in vivo and in vitro. Therefore, to obtain a thorough picture the glycosylation patterns should be investigated too. CONCLUSIONS In conclusion, our findings introduce the working hypothesis that RD VH is characterized by altered proteolysis of structural and non-structural components. Some of the findings here reported, in particular those supporting a proteolysis-based alteration of angiogenic processes may represent a molecular rationale for the morphological and functional abnormalities of vessels microarchitecture RD subjects have been reported to develop. Conceptually, it is very difficult to speculate whether the alterations observed contribute to RD onset or are a consequence of the disease. In this last case, it may be relevant to investigate whether they predispose to recurrent RD and PVR. Therefore, further studies on a larger cohort of samples and using additional enrichment strategies are demanded to clarify the pathogenic role of the proteolytic alterations here identified. We nonetheless believe that the approach here undertaken has the merit to pose a scientific question that may foster novel pathogenetic perspectives on RD pathobiology and, hopefully, new therapeutic targets and potential biomarkers to predict the recurrence of RD and/or PVR. Abbreviations AH Aqueous Humor BCA Bicinchoninic acid assay BP Biological Process CART Classification and regression trees CC Cellular Component DDA Data Dependent Acquisition DEP Differentially Expressed Protein DTT Dithiothreitol EMT Epithelial to Mesenchymal Transformation GO Gene Ontology ILM Inner Limiting Membrane LFQ Label Free Quantification MF Molecular Function OPN Osteopontin PCA Principal Component Analysis PD Proteome Discoverer PPV Pars Plana Vitrectomy PTM Post-Translational Modification PVR Proliferative Vitreo-Retinopathy RD Retinal Detachment RP Retinitis Pigmentosa RPE Retinal Pigment Epithelium RRD Recurrent RD SEA Sperm protein, Enterokinase and Agrin TFA Trifluoroacetic acid Ub Ubiquitin UHPLC Ultra High Performance Liquid Chromatography VH Vitreous Humor VTN Vitronectin Declarations CONFLICT OF INTEREST The authors declare that do not have pending conflict of interest with the study reported SUPPORTING DATA Data presented in this manuscript are supported by additional supporting information. Supplementary Figure and Table files have been uploaded. FUNDING INFORMATION The authors further received financial support from LazioInnova (grant: A0375-2020-36591). This study was supported by Next Generation Promising (NGP) and PRIN-MIUR (grant: 2022R9WCZS_001). Author Contribution G.A.Z.: conceptualization, data curation, formal analysis, investigation, methodology, software, writing; S.G.: conceptualization, data curation, formal analysis, software, writing; I.P.: data curation, formal analysis; L.P., G.R., G.R.T., G.G., A.B.: conceptualization, formal analysis, supervision and validation; P.A.B.: conceptualization, data curation, investigation, methodology; T.R.: writing – review and editing, supervision, resources; D.S.: conceptualization, data curation, investigation, writing – review and editing, supervision, resources. Acknowledgement The authors acknowledge the Ministry of Health and Fondazione Roma for the support. G.A. Zingale and I. 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Supplementary Files SupplementaryFiguresSubmission.docx SupplementaryTablesSubmission.docx Cite Share Download PDF Status: Published Journal Publication published 14 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 23 May, 2025 Reviews received at journal 26 Nov, 2024 Reviewers agreed at journal 12 Nov, 2024 Reviewers agreed at journal 11 Nov, 2024 Reviewers invited by journal 11 Nov, 2024 Editor assigned by journal 11 Nov, 2024 Editor invited by journal 07 Nov, 2024 Submission checks completed at journal 05 Nov, 2024 First submitted to journal 05 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5395071","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":377357696,"identity":"d5322fde-a099-4066-859e-8b4baf524e8b","order_by":0,"name":"Diego Sbardella","email":"data:image/png;base64,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","orcid":"","institution":"IRCCS Fondazione Bietti","correspondingAuthor":true,"prefix":"","firstName":"Diego","middleName":"","lastName":"Sbardella","suffix":""},{"id":377357697,"identity":"425d8fcb-bf79-44fa-a5cf-d116481a849d","order_by":1,"name":"Gabriele Antonio Zingale","email":"","orcid":"","institution":"IRCCS Fondazione Bietti","correspondingAuthor":false,"prefix":"","firstName":"Gabriele","middleName":"Antonio","lastName":"Zingale","suffix":""},{"id":377357698,"identity":"a3f513bd-ae65-4cb8-8077-aa36791b43bd","order_by":2,"name":"Sara Giammaria","email":"","orcid":"","institution":"IRCCS Fondazione Bietti","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Giammaria","suffix":""},{"id":377357699,"identity":"231bc6a5-9c31-4b9b-aea9-ca8367c51673","order_by":3,"name":"Irene Pandino","email":"","orcid":"","institution":"IRCCS Fondazione Bietti","correspondingAuthor":false,"prefix":"","firstName":"Irene","middleName":"","lastName":"Pandino","suffix":""},{"id":377357700,"identity":"dbfd7982-0d6c-44f5-afc8-e057efc529b3","order_by":4,"name":"Luca Placentino","email":"","orcid":"","institution":"IRCCS Fondazione Bietti","correspondingAuthor":false,"prefix":"","firstName":"Luca","middleName":"","lastName":"Placentino","suffix":""},{"id":377357701,"identity":"688feb32-3277-4a73-bb28-c8226607bc49","order_by":5,"name":"Guido Ripandelli","email":"","orcid":"","institution":"IRCCS Fondazione Bietti","correspondingAuthor":false,"prefix":"","firstName":"Guido","middleName":"","lastName":"Ripandelli","suffix":""},{"id":377357702,"identity":"57e47914-14d9-4a93-a0d7-5004e8ceca60","order_by":6,"name":"Grazia Raffaella Tundo","email":"","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Grazia","middleName":"Raffaella","lastName":"Tundo","suffix":""},{"id":377357703,"identity":"0f807852-c0e1-4ff7-b530-f967a652b2a5","order_by":7,"name":"Giuseppe Grasso","email":"","orcid":"","institution":"University of Catania","correspondingAuthor":false,"prefix":"","firstName":"Giuseppe","middleName":"","lastName":"Grasso","suffix":""},{"id":377357704,"identity":"89c081d8-8a1f-46da-97ac-320d70094bc7","order_by":8,"name":"Alessio Bocedi","email":"","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Alessio","middleName":"","lastName":"Bocedi","suffix":""},{"id":377357705,"identity":"6312dc9e-91c6-4b6e-879d-0f14b439618c","order_by":9,"name":"Peter Bell","email":"","orcid":"","institution":"University of British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Bell","suffix":""},{"id":377357706,"identity":"03ea0fc7-2e45-4b87-96a8-503db34de354","order_by":10,"name":"Tommaso Rossi","email":"","orcid":"","institution":"IRCCS Fondazione Bietti","correspondingAuthor":false,"prefix":"","firstName":"Tommaso","middleName":"","lastName":"Rossi","suffix":""}],"badges":[],"createdAt":"2024-11-05 11:38:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5395071/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5395071/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-19857-z","type":"published","date":"2025-10-14T15:57:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70404270,"identity":"76478ebc-0d86-4918-a15c-67acca1b4f83","added_by":"auto","created_at":"2024-12-02 22:59:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90926,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the experimental design and study workflow. Created with icons from BioRender.com.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5395071/v1/ad664b563b3f673bc2544f55.png"},{"id":70404272,"identity":"cbf4b1d9-cc43-42ac-a382-1342e35fe0cf","added_by":"auto","created_at":"2024-12-02 22:59:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98592,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Venn diagram showing the number of proteins identified, either common to RD and Ctrl, or specific to one of the two experimental groups; (B) Density plot of log\u003csub\u003e2\u003c/sub\u003e transformed protein intensities without applying quantile normalization; (C) Density plot of log\u003csub\u003e2\u003c/sub\u003e transformed protein intensities after application of class-specific quantile normalization; (D) PCA analysis of Ctrl (yellow) and RD (blue) proteins, PC1 and PC2 % values are reported. Centroid values are reported for both groups.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5395071/v1/b07ba08bcf885426e743d3d1.png"},{"id":70404269,"identity":"d7aa2d38-99c1-4fb3-b968-26c266e5654d","added_by":"auto","created_at":"2024-12-02 22:59:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30067,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano plot showing DEPs. X-axis reports the fold change expressed as log\u003csub\u003e2\u003c/sub\u003e fold-change (Log\u003csub\u003e2\u003c/sub\u003eFC); Y-axis reported the -log\u003csub\u003e10\u003c/sub\u003e p-value adjusted by Benjamini-Hochberg correction (p-value\u003csub\u003eadj\u003c/sub\u003e). Statistical significance was set for p.ord≤0.05. Dashed lines highlighted the cut-off set for log\u003csub\u003e2\u003c/sub\u003eFC and log\u003csub\u003e10 \u003c/sub\u003eof p-value significance, namely: ±0.57 and 1.3, respectively. Proteins upregulated and downregulated in RD vs Ctrl VH are represented in red and turquoise, respectively.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5395071/v1/d2282da267f62b014ab4d19e.png"},{"id":70404271,"identity":"9d0e08dc-a713-4535-be7b-597f151e888f","added_by":"auto","created_at":"2024-12-02 22:59:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":292061,"visible":true,"origin":"","legend":"\u003cp\u003eMF chart showing the terms enriched by submitting proteins upregulated or exclusive of Ctrl (A) and RD (B) VH to GO. GeneRatio, which corresponds to the proportion of genes the dataset annotated with GOTERM, was calculated and data filtered for p≤0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5395071/v1/2424e455516ab43d2ed2c74b.png"},{"id":70404277,"identity":"58011549-fd47-43eb-9806-90e6dd9eba5f","added_by":"auto","created_at":"2024-12-02 22:59:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":145934,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Venn diagrams showing the distribution of N-termini identified in Ctrl and RD VH; Sequence logo plot generated by analyzing the aminoacidic preferences of neo N-termini of Ctrl (B) and RD (C). Dashed line (P1, P1’ position) indicates the cleavage site. (D) Venn diagrams showing the distribution of C-termini identified in Ctrl and RD VH.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5395071/v1/4b1a448d388542fa1fe4b952.png"},{"id":70404275,"identity":"b25312cb-b52c-4a16-879d-d73e54d93200","added_by":"auto","created_at":"2024-12-02 22:59:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":237443,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the main N-terminal and C-terminal cleavage sites identified in this study.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5395071/v1/e6df0f0220aac4196e1800af.png"},{"id":70404278,"identity":"6daa7282-7617-47b1-880b-f1ab603c6622","added_by":"auto","created_at":"2024-12-02 22:59:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":499910,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blotting panel showing a representative immunostaining of a panel of VH proteins of interest, namely: OPN, VTN, Interphotoreceptor Matrix Proteoglycans 1 and 2 (IMPG-1 and -2, respectively), DKK3.\u003c/p\u003e\n\u003cp\u003eBlack arrows indicate putative proteolytic fragments of the protein of interest. Asterisks indicate non-specific bands.\u003c/p\u003e","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5395071/v1/2abad76e8ee7dd6b4510ecfb.png"},{"id":93955983,"identity":"0f836a36-569c-4c58-b247-5ae1bf385258","added_by":"auto","created_at":"2025-10-20 16:08:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3254992,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5395071/v1/e557c10d-3d58-4e3b-ac3c-3cb5d795bcfe.pdf"},{"id":70404961,"identity":"e7c68b7c-0e8f-47e7-b03a-2478ae6893c7","added_by":"auto","created_at":"2024-12-02 23:07:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":403626,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresSubmission.docx","url":"https://assets-eu.researchsquare.com/files/rs-5395071/v1/2eac574a890043c3157b9861.docx"},{"id":70404960,"identity":"a0d43218-576d-4b79-9427-8d315b096fed","added_by":"auto","created_at":"2024-12-02 23:07:57","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":196331,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablesSubmission.docx","url":"https://assets-eu.researchsquare.com/files/rs-5395071/v1/354f2ca7a44145156ad83d33.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAltered Pattern of Proteolysis of Extracellular Proteins in Rhegmatogenous Retinal Detachment by Mining of Tryptic and N-Termini Datasets From Vitreous Humor Proteome\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRhegmatogenous Retinal Detachment (RD) is a severe condition characterized by the detachment of the neurosensory retina from the Retinal Pigment Epithelium (RPE) due to the presence of retinal tears\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The incidence of RD is 7 to 12/10.000 cases per year\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e with a guarded prognosis and a heavy burden for the patients\u0026rsquo; quality of life and health care expenditures \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Recurrent RD occurs in up to 20% of cases \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, leading to severe visual loss and often requiring multiple surgery\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eProliferative Vitreo-Retinopathy (PVR)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e is the leading cause of Recurrent RD (RRD) and of severe visual loss, and is characterized by the growth and contraction of cellular aggregates on the retinal surface and within the vitreous chamber\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The Epithelial to Mesenchymal Transformation (EMT) represents the hallmark of PVR and comprises the acquisition of morphological and functional properties of RPE cells, resulting in blood hemodynamics alterations, vessel microarchitecture changes, fibrosis and, eventually, retinal contraction \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe molecular footprint and the cascade of events promoting EMT and the pathological retinal remodeling associated to PVR has been extensively studied, yet is still far to be fully understood. Previous studies have shown changes in RPE and photoreceptors metabolism, cell adhesiveness and polarization, extracellular matrix turnover and remodeling, immune system cross-talk, redox unbalance, angiogenesis and inflammation\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePars Plana Vitrectomy (PPV) is the elective surgical procedure for complex RDs and allows the collection of vitreous (VH) and aqueous (AH) humor samples. Research activities on these fluids are intrinsically complex since protein concentration is typically very low and identification of relevant proteins is hidden by abundant contaminating proteins (e.g., albumin, IgG etc.), often requiring sample pooling procedures and off-line separation or depletion of these proteins.\u003c/p\u003e \u003cp\u003ePrevious relevant proteomics studies applied to the human vitreous and specifically to RD patients suggested novel molecular perspectives on RD and PVR pathobiology\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, to date, post-translational modification (PTM) profiling of VH samples has not yet (to the best of our knowledge) been performed. PTMs such as phosphorylation, glycosylation, and proteolytic processing are able to profoundly alter the structure and function of a protein\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In this regard, we reasoned that retinal detachment could be linked to the dysregulation of physiological PTMs and, in particular, endogenous proteolytic events carried out by the different classes of enzymes populating the VH.\u003c/p\u003e \u003cp\u003eThe present study reports the results of a pilot shotgun Label Free Quantification (LFQ) proteomic characterization of vitreous samples collected from primary RD patients (n\u0026thinsp;=\u0026thinsp;8), compared to controls (n\u0026thinsp;=\u0026thinsp;8) who underwent vitreoretinal surgery for formation of epiretinal membranes. Without immunodepleting for most abundant proteins and working on a single-subject scale, we identified a total 79 differentially expressed proteins (DEPs) between RD and Ctrl VH, uncovering precise alterations of key pathways for cell metabolism and tissue homeostasis. Thereafter, using proteomics technologies and analysis tools,\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e we explored the repertoire of endogenous N-termini (and C-termini). This approach identified several known and unknown sites of proteolysis in structural and non-structural VH components and significant alterations on proteins serving roles for immune system regulation, proteolytic balance and, in particular, angiogenesis. This scenario introduces the perspective that the pathogenesis of RD progresses through unbalanced extracellular proteolysis.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics Approval \u0026ndash; Study Design\u003c/h2\u003e \u003cp\u003eThe study was approved by the local ethics committee (P.R.O. Project, 09/14/2023). The main goal of the study is to collect vitreous and aqueous humour to investigate pathogenic determinants and potential biomarkers of most prevalent retinal disorders, including retinal detachment, by proteomics approaches.\u003c/p\u003e \u003cp\u003eSamples collected are stored in an internal biobank of the institute.\u003c/p\u003e \u003cp\u003eIn accordance to the tenets of the Declaration of Helsinki, enrolled patients were asked to read and sign an informed consent form.\u003c/p\u003e \u003cp\u003eInclusion criteria for cases: age\u0026thinsp;\u0026gt;\u0026thinsp;18 years, primary rhegmatogenous retinal detachment who underwent vitrectomy, phakic or pseudophakic patients.\u003c/p\u003e \u003cp\u003eInclusion criteria for controls: age\u0026thinsp;\u0026gt;\u0026thinsp;18 years, patients who underwent vitrectomy for reasons different from retinal detachment (e.g., epiretinal membranes, dislocated intraocular lens), phakic or pseudophakic patients.\u003c/p\u003e \u003cp\u003eExclusion criteria (apply to both experimental groups): diabetes, collagenopathies (e.g. Ehlers-Danlos Syndrome), cancer, vitreous hemorrhages, concomitant neurodegenerative, inflammatory or infective eye diseases, alterations of the electrophoretic profile of the gamma-globulin band\u003c/p\u003e \u003cp\u003eVitreous samples were obtained from patients undergoing PPV for either retinal detachment or epiretinal membrane peeling. Approximately 1 mL of fluid were collected using a sterile 5 mL syringe and poured into a sterile 1.7 mL Eppendorf test tube. Samples were then immediately cleared by centrifugation and protein concentration quantified by bicinchoninic acid assay (BCA). Thereafter, samples were subdivided into different aliquots and stored at -80\u0026deg;C until use. No freeze/thaw cycles were applied for proteomics or Western blotting studies.\u003c/p\u003e \u003cp\u003eAll samples discussed in the study were processed and analysed in parallel. Anonymity of subjects was guaranteed throughout the entire study.\u003c/p\u003e \u003cp\u003eInitially, n\u0026thinsp;=\u0026thinsp;9 RRD cases and controls were enrolled in the study and analysed by mass spectrometry. One subject per group was then filtered out from analysis for technical issues during sample preparation. Raw mass spectrometry data of these samples were uploaded (corresponding to original samples #6 and #16) in the PRIDE repository (see \u0026ldquo;Data availability statement\u0026rdquo;) together with all samples effectively analysed and part of the study.\u003c/p\u003e \u003cp\u003eSelection of samples was based on the best matching of demographic and clinical characteristics of the subjects. These data are introduced in the \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003eresults\u003c/span\u003e section and further reported in Table I.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMass spectrometry\u003c/h3\u003e\n\u003cp\u003eIn the case of proteomic analysis, samples (100 \u0026micro;g of VH proteins) were dehydrated in a SpeedVacuum system, reconstituted in denaturing buffer (6M guanidine-HCl, 50 mM Hepes, pH 7.8).\u003c/p\u003e \u003cp\u003eProteins were then reduced [5 mM dithiothreitol, 45 min, room temperature (r.t.)], alkylated (10 mM iodoacetamide, 30 min, r.t) and digested with trypsin (1:50 enzyme:protein ratio, overnight, 37\u0026deg;C) (Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e \u003cp\u003eTrypsin digestion was quenched with 0.4% trifluoroacetic acid (TFA) and peptides cleaned using Stage-Tips (C18 resin) (Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e \u003cp\u003eThereafter, eluted peptides were dried by SpeedVacuum system and resuspended in 2% Acetonitrile, 0.05% Trifluoracetic Acid for mass spectrometry injection.\u003c/p\u003e \u003cp\u003eProteomic analysis was performed injecting (twice) 1 \u0026micro;g peptides for each experimental conditions into an Orbitrap Exploris 240 mass spectrometer coupled to an Ultimate 3000 nano-ultra high performance liquid chromatography (nano-UHPLC) system. Solvent A: 100% H\u003csub\u003e2\u003c/sub\u003eO, 0.1% Formic Acid; Solvent B: 80% Acetonitrile, 0.1% Formic Acid. UHPLC Gradient (minutes - %B): 0\u0026ndash;6.7; 2\u0026ndash;6.7; 62\u0026ndash;34.4; 67\u0026ndash;55.5; 72\u0026ndash;100; 80\u0026ndash;100; 82\u0026thinsp;\u0026minus;\u0026thinsp;6.7; 88\u0026thinsp;\u0026minus;\u0026thinsp;6.7. Column oven temperature: 45\u0026deg;C. Run time: 88 min. Loading Pump flowrate: 30 \u0026micro;L/min. NC Pump flowrate: 250 nL/min. Data acquisition was conducted in Data Dependent Acquisition (DDA) mode.\u003c/p\u003e \u003cp\u003eOrbitrap Resolution: 120,000; Scan Range (m/z): 375\u0026ndash;1650; RF Lens (%): 80; Normalized AGC Target (%): 300. ddMS\u0026sup2; was triggered using the following filters: Isolation Window (m/z): 2; Normalized HCD Collision Energy (%): 30; Orbitrap Resolution: 15000; Normalized AGC Target (%): 50.\u003c/p\u003e\n\u003ch3\u003eData and Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eProtein and peptides were searched using Proteome Discoverer (PD) software (v. 2.5, Thermo Fisher Scientific) against a UniProt human protein FASTA database including protein isoforms. Sequest implemented with the Inferys rescoring algorithm was used and a concatenated target-decoy strategy applied for determination of the proteins False Discovery Rate (strict FDR\u0026thinsp;\u0026le;\u0026thinsp;0.01 and relaxed FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eTrypsin (full) was set as enzyme, 10 ppm precursor mass tolerance and 0.02 Da fragment mass tolerance. Carbamidomethylation of cysteines (+\u0026thinsp;57.021) was set as static modification, whereas oxidation on methionine (+\u0026thinsp;15.995) as dynamic modification per peptides according to the requirements of Inferys algorithm.\u003c/p\u003e \u003cp\u003eThe search of N-termini and C-termini was instead run using FragPipe/MsFragger (v. 22.0), as reported elsewhere\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe resulting protein and peptides identifications and quantitation data were analysed using an in-house built R script (v. 2.5). In-depth statistical analysis for differential protein expression between RD and control groups are described throughout the \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003eresults\u003c/span\u003e section.\u003c/p\u003e\n\u003ch3\u003eWestern blotting studies\u003c/h3\u003e\n\u003cp\u003eA selection of proteins identified by the proteomic approach was further analyzed by denaturing and reducing Western blotting using an aliquot of VH not denatured by guanidine-HCl as described before.\u003c/p\u003e \u003cp\u003eIn all cases, 5 \u0026micro;g of vitreous proteins per subject were heat-denatured and reduced in Laemmli buffer 1x supplemented with 1 mM dithiothreitol (DTT). Thereafter, 4\u0026ndash;20% acrylamide pre-cast gels (Bio-Rad, Hercules, CA, USA) were used to separate proteins by SDS-PAGE. After separation, proteins were transferred to a HyBond-ECL nitrocellulose filters (Bio-Rad, Hercules, CA, USA) and probed with the antibodies indicated. All antibodies used were purchased from ProteinTech (Rosemont, Illinois, USA). Antibodies were diluted 1:3000 in 0.1% Tween-PBS 0.1% fat-free milk and with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody (Bio-Rad, Hercules, CA, USA), diluted 1:10.000 in 0.1% Tween-PBS 0.1% fat-free milk.\u003c/p\u003e \u003cp\u003eProteins were developed by ECL chemiluminescence and recorded in a iBright 1500 (ThermoFisher scientific).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSubjects enrollment and study design\u003c/h2\u003e \u003cp\u003eVitreous samples were obtained from patients undergoing PPV for either RD (RD, n\u0026thinsp;=\u0026thinsp;8, mean age 69\u0026thinsp;\u0026plusmn;\u0026thinsp;4 years) or macular pucker (n\u0026thinsp;=\u0026thinsp;8, mean age 75\u0026thinsp;\u0026plusmn;\u0026thinsp;6 years), enrolled as controls (Ctrl). Additional demographic, epidemiological and clinical data of enrolled patients are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Systemic hypertension was the prevalent co-morbidity across enrolled subjects. A heterogeneous localization (supero-temporal and inferior) of the retinal detachment was documented in the RD group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and clinical parameters of subjects\u0026rsquo; enrolled in the study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAge*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eComorbidity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSystemic Therapy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVR Disease\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEye\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eVisual Acuity**\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePseudophakia\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD #1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSystemic Hypertension, Gilbert syndrome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ-blocker, Ca\u003csup\u003e2+\u003c/sup\u003e channel blocker, ARB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSupero-Temporal RD, Vitreal Proliferation Signs, Macula Off\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMotu Manu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD #2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInfero-Temporal RD, Vitreal Proliferation Signs, Macula Off\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMotu Manu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD #3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSystemic Hypertension, myocardial infarction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSartan, β-blocker, Xa Factor inhibitor, Antiarrhythmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSupero-Temporal RD, Vitreal Proliferation Signs, Macula Off\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLight Perception\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD #4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSystemic Hypertension\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ-blocker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSupero-Temporal RD, Vitreal Proliferation Signs, Macula Off\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLight Perception\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD #5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSystemic Hypertension, Osteoporosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHydrochlorothiazide\u0026thinsp;+\u0026thinsp;sartan, Vitamin D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSubtotal RD, Macula Off\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD #6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSystemic Hypertension, myocardial infarction, deep vein thrombosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ-blocker, Xa Factor inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInfero-Temporal RD, Macula On\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD #7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInferior RD, Macula Off\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMotu Manu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD #8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSystemic Hypertension\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ-blocker, ACE Inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInferior RD, Vitreal Proliferation Signs, Macula Off\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMotu Manu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTRL #1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSystemic Hypertension, Dysthyroidism, Hypercholesterolemia, Benign prostatic hypertrophy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ-blocker, levothyroxine, 5 alpha reductase inhibitor, Statin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEpiretinal Membrane, Cataract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTRL #2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEpiretinal Membrane, Cataract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTRL #3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEpiretinal Membrane, Cataract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTRL #4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSystemic Hypertension, Atrial fibrillation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProton pump inhibitor, β-blocker, ACE inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDislocated IOL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTRL #5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEpiretinal Membrane, Cataract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTRL #6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSystemic Hypertension, Hypercholesterolemia, Osteoporosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHydrochlorothiazide\u0026thinsp;+\u0026thinsp;Sartan,Statin, Vitamin D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMacular Hole, Epiretinal Membrane, Cataract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTRL #7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMacular Hole, Epiretinal Membrane, Cataract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTRL #8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEpiretinal Membrane, Cataract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e*Years RD\u0026thinsp;=\u0026thinsp;Retinal Detachment\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e**Decimals IOL\u0026thinsp;=\u0026thinsp;Intraocular lens\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAn equal quantity (\u0026micro;g) of VH protein for each enrolled sample was digested with trypsin and subjected to proteomics analysis. A workflow of the study is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDysregulation of metabolic pathways and structural components of the ECM in VH from RD patients\u003c/h3\u003e\n\u003cp\u003eIdentification by MS yielded a total 2,134 proteins with \u0026ge;\u0026thinsp;1 unique peptides together with 7,162 peptide groups and 287,667 PSMs. Results were then filtered for Master proteins, as they are defined in PD glossary.\u003c/p\u003e \u003cp\u003eIn total 798 proteins were identified, with a robust overlap between RD and Ctrl: 745 proteins were common to the two experimental groups, 22 proteins were documented as exclusive of RD group and 31 of the Ctrl group (discussion of exclusive proteins is limited to those identified in \u0026ge;\u0026thinsp;50% subjects/group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In this regard, supplementary Tables\u0026nbsp;1 and 2 report the list of proteins exclusive for RD and Ctrl groups, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe distribution of protein intensities was verified upon log\u003csub\u003e2\u003c/sub\u003e transformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Density-plots of non-normalized intensities, though highlighting moderate intragroup variability, suggested a comparable intra-group and inter-group normal distribution for both RD and Ctrl datasets. Therefore a class-specific quantile normalization strategy was deemed applicable\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The experimental variability improved, as shown in the post-quantile normalization density plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Thereafter, missing values were imputed using Classification and regression trees (CART) approach\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eData were then analyzed by Principal Component Analysis (PCA) to further check for global differences between RD and Ctrl experimental groups; data distribution and the position of the group-specific centroid suggested that the biological data had indeed different features (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eDEPs between RD and Ctrl subjects were then analyzed using a moderate Bayesian t test LIMMA statistical approach and setting as threshold for significance log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026le;\u0026thinsp;0.57 and p\u0026thinsp;\u0026le;\u0026thinsp;0.05 adjusted by Benjamini-Hochberg correction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn total, 43 proteins were found as upregulated and 36 as downregulated in the RD group, compared to Ctrl (DR/Ctrl ratio); the entire list of upregulated and downregulated proteins in RD and Ctrl VH is shown in Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, respectively. To better cluster and rationalize data, DEPs discussed above and group-exclusive proteins identified and quantified were submitted to gene ontology (GO) to infer statistically enriched (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) molecular function (MF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B), cellular components (CC) (Suppl. Figure\u0026nbsp;1), and biological processes (BP) (Suppl. Figure\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProteins upregulated in RD VH. The table shows the Accession number (UniProt), the log2FC calculated for the RD/Ctrl ratio, the p.ordinary (p.ord) value calculated by applying the LIMMA test, the Protein Description, the number of peptides identified and the calculated FDR.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003elogFC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.ord\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e# Peptides\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFDR\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP10745\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.639\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03291\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRetinol-binding protein 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE7EUF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEctonucleotide pyrophosphatase/phosphodiesterase family member 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP01011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01991\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-1-antichymotrypsin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA0A1B0GWE8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.508\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCathepsin D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP10523\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS-arrestin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP36222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.473\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChitinase-3-like protein 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB4DPQ0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03967\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eComplement subcomponent C1r\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP02788\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.444\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLactotransferrin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ17R60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.445\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInterphotoreceptor matrix proteoglycan 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP02766\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.727\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTransthyretin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA0A7I2V2D2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePlasma protease C1 inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP62873\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGuanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP02751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.082\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02407\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFibronectin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP01834\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.702\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eImmunoglobulin kappa constant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP08571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMonocyte differentiation antigen CD14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ15582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04921\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTransforming growth factor-beta-induced protein ig-h3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP10451\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOsteopontin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP07858\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02937\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCathepsin B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ9BZV3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.318\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00486\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInterphotoreceptor matrix proteoglycan 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP69905\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.387\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHemoglobin subunit alpha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP01033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.669\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMetalloproteinase inhibitor 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ6EMK4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.303\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVasorin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ92743\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04816\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerine protease HTRA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP07602\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01956\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProsaposin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP61626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.947\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLysozyme C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP04040\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04360\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP05546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHeparin cofactor 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ96JP9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00677\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCadherin-related family member 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP55058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.941\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePhospholipid transfer protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP01619\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eImmunoglobulin kappa variable 3\u0026ndash;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ9HCQ7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.576\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00492\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePro-FMRFamide-related neuropeptide VF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ8N114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.473\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein shisa-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH0YCV9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.792\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCD44 antigen (Fragment)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ9BYJ0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.665\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02877\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFibroblast growth factor-binding protein 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP02747\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eComplement C1q subcomponent subunit C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA0A0C4DH73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eImmunoglobulin kappa variable 1\u0026ndash;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP17936\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01349\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInsulin-like growth factor-binding protein 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ9NP84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03531\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTumor necrosis factor receptor superfamily member 12A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP84243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.906\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01618\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHistone H3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ8NA57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUncharacterized protein C12orf50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP81172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.653\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHepcidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJ3KQ66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02705\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReelin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP10586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.624\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReceptor-type tyrosine-protein phosphatase F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProteins downregulated in RD VH. The table shows the Accession number (UniProt), the log\u003csub\u003e2\u003c/sub\u003eFC calculated for the RD/Ctrl ratio, the p.ord value calculated by LIMMA test, the Protein Description, the number of peptides identified and the calculated FDR.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003elogFC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.ord\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e# Peptides\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFDR\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP53674\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.506\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-crystallin B1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP00352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01493\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAldehyde dehydrogenase 1A1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP43320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04370\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-crystallin B2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP48637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlutathione synthetase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP22914\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04731\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGamma-crystallin S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP05813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.910\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-crystallin A3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP29401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.471\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTransketolase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP26998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02451\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-crystallin B3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP53673\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.784\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02749\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-crystallin A4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ00796\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.797\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSorbitol dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP53672\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-4.941\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00722\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-crystallin A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP16152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-4.295\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03914\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCarbonyl reductase [NADPH] 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ01082\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.667\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSpectrin beta chain, non-erythrocytic 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP04792\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.959\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00299\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHeat shock protein beta-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ71U36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04348\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTubulin alpha-1A chain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP09936\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-4.167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUbiquitin carboxyl-terminal hydrolase isozyme L1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ93088\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00427\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBetaine\u0026ndash;homocysteine S-methyltransferase 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ96GW7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.696\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrevican core protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP15121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.489\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAldo-keto reductase family 1 member B1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP07900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.368\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHeat shock protein HSP 90-alpha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP22061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.692\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein-L-isoaspartate(D-aspartate) O-methyltransferase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP14174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00727\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMacrophage migration inhibitory factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP22392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.535\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNucleoside diphosphate kinase B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP63104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02565\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14-3-3 protein zeta/delta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA0A8Q3WKK9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.820\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLactase-like protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ9NS15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02936\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLatent-transforming growth factor beta-binding protein 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ16555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.719\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00993\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDihydropyrimidinase-related protein 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ14019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.415\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCoactosin-like protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB5MDF5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.498\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04477\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGTP-binding nuclear protein Ran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ05639\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.494\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eElongation factor 1-alpha 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE9PQW4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMitogen-activated protein kinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD6RA82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-5.687\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnnexin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH7C4I3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-5.215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRuvB-like helicase (Fragment)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ86SR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-4.559\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03738\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePolypeptide N-acetylgalactosaminyltransferase 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP37837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTransaldolase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP03973\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03092\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAntileukoproteinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWith regard to proteins upregulated in RD VH, it is worth mentioning extracellular matrix components such as interphotoreceptor matrix proteoglycan 1 and 2 (Q17R60 and Q9BZV3, respectively) and fibronectin (P02751). Moreover, proteins involved in TGF-β signaling and pro-inflammatory cascades or angiogenic processes such as transforming growth factor-beta-induced protein Ig-h (Q15582), fibroblast growth factor binding protein (Q9BYJ0), vasorin (Q6EMK4) and osteopontin (P10451) were found upregulate together with complement factors and natural inhibitors of peptidase and protease activity, such as metalloproteinase inhibitor 1 (P01033), α1-antichymotrypsin (P01011) and heparin cofactor 2 (P05546).\u003c/p\u003e \u003cp\u003eAccordingly, GO term analysis identified enrichment (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) of the following terms: collagen-containing extracellular matrix (GO:006023), photoreceptor outer segment and cell cilium (GO:0001750 and GO:0097733, respectively), laminin (GO:0043236) and fibronectin binding (GO:0001968), enzyme inhibitor activity (GO:0004857) peptidase inhibitor and endopeptidase regulatory activity (GO:0030414 and GO:0061135, respectively).\u003c/p\u003e \u003cp\u003eConversely, proteins downregulated in RD VH were mostly involved in proteostasis regulation and energetic/metabolic cycles, including carbonyl reductase (P16152), transketolase and transaldolase (P29401 and P37837, respectively), glutathione synthetase and reductase (P48637 and P00390, respectively), stress proteins, such as heat shock protein 90 (P07900), β-crystallins A3 and B1 (P05813, P53674, respectively) and proteins involved in ubiquitin (Ub) signaling, such as lengsin (Q5TDP6), ubiquitin carboxyl-terminal hydrolase isozyme L1 (P09936).\u003c/p\u003e \u003cp\u003eAccordingly, GO charts highlighted an enrichment in terms referable to mechanisms of proteostasis regulation and energetic metabolism such as NAD binding, (GO:0051287), protein folding chaperone (GO:0044183), oxidoreductase activities (GO:0016616, GO:0016614),\u003c/p\u003e\n\u003ch3\u003eMining of N-termini highlights dysregulation of proteolysis in vitreous humor from RD patients\u003c/h3\u003e\n\u003cp\u003eGiven the enrichment of peptidases and inhibitors that was observed in functional enrichment analysis of DEPs, we hypothesized that RD patients may suffer from unbalanced extracellular proteolysis. To test this hypothesis, FragPipe/MSFragger was used to search for semi-tryptic peptides indicative of proteolytic processing, with the specific aim of identifying N-termini and C-termini. A peptide-level FDR\u0026thinsp;\u0026le;\u0026thinsp;0.01 was applied, and the main criteria for N-termini identification were set for the absence of a lysine (K) or arginine (R) residue at the N-terminal flanking position of the peptide. This last search parameters were set to filter out peptides probably generated by the trypsin digestion of samples, thus not representing \u003cem\u003ebona fide\u003c/em\u003e endogenous N-termini.\u003c/p\u003e \u003cp\u003eThe search parameters chosen for the analysis enabled the identification of two classes of termini: mature N-termini, which, in most cases, originate from the enzymatic processing (often intracellular) of the signal peptide during protein secretion, and neo N-termini, which typically correspond to enzymatic cleavage of mature proteins.\u003c/p\u003e \u003cp\u003eUpon further filtering for N-termini identified in at least 3 out of 8 subjects/group, the search identified a total of 457 peptides common to RD and Ctrl VH, 182 exclusive of RD and 216 of Ctrl VH samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Neo N-termini were more represented than mature N-termini in both experimental groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eN-termini documented as exclusive of either one of the two groups, and N-termini upregulated or downregulated (LIMMA test, Supplementary Fig.\u0026nbsp;3) were submitted (separately for each group) to TopFINDER (v. 4.1, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://topfind.clip.msl.ubc.ca/\u003c/span\u003e\u003cspan address=\"https://topfind.clip.msl.ubc.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to search for known cleavage sites of proteins identified and putative enrichment of specific proteases (the complete name of enzyme discussed below is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLists of enzymes and their abbreviations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcronym\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnzyme Name\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP2, MMP9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMatrix Metalloproteinase 2, 9 (Gelatinases)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP3, MMP7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMatrix Metalloproteinase 3, 7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMatrix Metalloproteinase 12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBone Morphogenic Protein 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCATH-D, -S, -L, -B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCathepsin-D, -S, -L, -B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKLK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKallikrein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMEP1B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeprin 1B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADAMTS-10, -17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA Disintegrin and Metalloproteinase with Thrombospondin motifs-10, -17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBACE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBeta-secretase 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA separated discussion for N-termini enriched/exclusive of either Ctrl or RD subjects is then provided for the sake of the readership.\u003c/p\u003e \u003cp\u003eSeveral previously characterized neo N-termini were identified as downregulated (-0.57 Log\u003csub\u003e2\u003c/sub\u003eFC, p\u003csub\u003eadj\u003c/sub\u003e\u0026le;0.05) in the RD/Ctrl ratio or specific (assigned for those identified and quantified in \u0026ge;\u0026thinsp;75% subjects) of Ctrl VH samples (Supplementary Table\u0026nbsp;3). They include: i) several (n\u0026thinsp;=\u0026thinsp;15) previously characterized neo-terminal fragments of crystallin αB released by MMP9; ii) a wide array of proteolytic fragments of additional crystallins, such as γ-crystallins (isoforms 2\u0026thinsp;\u0026minus;\u0026thinsp;1, 4 and S, P07315, P07320 and P22914, respectively), β-crystallin (P43320) and heat shock protein β4 (P02489); iii) a neo N-terminus of serpin-B9; iv) a previously unknown neo N-terminus of protein bassoon (Q9UPA5), which is a presynaptic protein and major component of photoreceptor ribbon. In this case, the potential cleavage site was mapped between \u003csup\u003e1228\u003c/sup\u003e\u0026darr;Tyr\u003csup\u003e1229\u003c/sup\u003e; v) a neo N-terminus of osteopontin (P10451-4). This fragment is generated by cleavage of the protein at \u003csup\u003e185\u003c/sup\u003e\u0026darr;Ala\u003csup\u003e186\u003c/sup\u003e by MMP3/MMP7\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterestingly, although the observation did not reach statistical significance, a previously unknown neo N-terminus of interphotoreceptor matrix proteoglycan 2 (IMPG2) (Q9BZV3) was found enriched in Ctrl VH (Supplementary Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eWith regard to mature N-termini enriched in Ctrl VH, it is worth documenting the identification of Wnt1 inhibitory factor (Q9Y5W5), secreted frizzled-related protein 3 (DKK3) (Q92765), spondin-1 (Q9HCB6), oligodendrocyte myelin glycoprotein (Q5SSB8), APP amyloid-beta precursor protein (P05067) and isoform 2 of fibrinogen alpha chain (A0A0S2Z3E8). In most cases, these findings were consistent with the tryptic dataset (Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eIn the case of VH samples of RD subjects, although a very limited number of upregulated N-termini (DR/Ctrl ratio, \u0026ge;\u0026thinsp;0.57 Log\u003csub\u003e2\u003c/sub\u003eFC, padj\u0026thinsp;\u0026le;\u0026thinsp;0.05) was identified, mature N-termini of several enzyme inhibitors and immune system components were documented (Supplementary Table\u0026nbsp;5): i) C3 and PZP-like alpha-2-macroglobulin domain-containing protein 5 (P01023); ii) α-1-antichymotrypsin (P0101); iii) complement factor B (P00751); iv) cystatin-3 (P01034); v) CSF1 receptor (P07333); vi) retinol binding protein (P10745); vii) insulin binding proteins-4 (P22692); vi) serum amyloid A protein (P0DJI8). In this last case, the peptide was previously reported to be released by the CATL1, MMP3 and CATB cut at residues \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u0026darr;Ser\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLooking beyond the neo-termini which were specific of RD VH group (focusing on those identified and quantified in \u0026ge;\u0026thinsp;50% subjects/group) highlighted several known and unknown peptides serving roles for the regulation of cell adhesiveness and, most notably, angiogenesis (Supplementary Table\u0026nbsp;5), yielding insights into the proteolytic landscape of vitreous humor: i) several proteolytic fragments of plasma proteins such as Albumin (P02768), including a previously unknown fragment generated by MEP1B and BACE2 cut between residues \u003csup\u003e174\u003c/sup\u003e\u0026darr;Ala\u003csup\u003e175\u003c/sup\u003e; ii) serotransferrin (P02787) identified by two legumain cleavage sites mapped between residue \u003csup\u003e601\u003c/sup\u003e\u0026darr;Asn\u003csup\u003e602\u003c/sup\u003e and \u003csup\u003e602\u003c/sup\u003e\u0026darr;Pro\u003csup\u003e603\u003c/sup\u003e; iii) a fragment of complement C1r subcomponent (P00736), with a cleavage site located between residues \u003csup\u003e186\u003c/sup\u003e\u0026darr;His\u003csup\u003e187\u003c/sup\u003e; iv) a fragment of clusterin (isoform 2, P10909-2) for which a cleavage fragment generated by MMP-3 and MMP12 at residues \u003csup\u003e309\u003c/sup\u003e\u0026darr;Met\u003csup\u003e310\u003c/sup\u003e was documented; v) calsyntenin-3 (isoform 2, Q9BQT9-2) reporting a cleavage site of ADAMTS-10 and \u0026minus;\u0026thinsp;17 at residues \u003csup\u003e824\u003c/sup\u003e\u0026darr;Val\u003csup\u003e825\u003c/sup\u003e; vi) apolipoprotein A1 showing a MMP7 and MMP12 cleavage site at residues \u003csup\u003e224\u003c/sup\u003e\u0026darr;Ser\u003csup\u003e225\u003c/sup\u003e; vii) fibronectin A - isoform2 for which two cleavage sites of MMP2, MMP8, MMP13 at residues \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e\u0026darr;Ala\u003csup\u003e20\u003c/sup\u003e and \u003csup\u003e20\u003c/sup\u003e\u0026darr;Asp\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e were documented; viii) basement membrane-specific heparan sulfate proteoglycan core protein (P98160). In this case, a peptide spanning across residue 4196\u0026ndash;4221 within the C-terminal portion of the molecule and a potential cleavage site between residues \u003csup\u003e4195\u003c/sup\u003e\u0026darr;Glu\u003csup\u003e4196\u003c/sup\u003e was identified. The fragment, called endorepellin, has been previously characterized to be released by BMP1 and to possess potent antiangiogenic activities\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e; ix) vitronectin (P04004), with a potential cleavage site not previously reported and located between residues \u003csup\u003e422\u003c/sup\u003e\u0026darr;Asp\u003csup\u003e423\u003c/sup\u003e and falling within the cell adhesion domain (hemopexin domain 4).\u003c/p\u003e \u003cp\u003eTo circumstantiate further these findings, cleavage sites derived from protein termini of RD and Ctrl groups were analyzed by a sequence logo plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,C).\u003c/p\u003e \u003cp\u003eA net prevalence of polar residues, in particular glycine (G) and serine (S) was observed in P1 and P1\u0026rsquo; position in the case of Ctrl VH (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eConversely, RD VH samples show a robust prevalence of polar residues such as alanine (A) and valine (V) over the same positions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThese preferences were confirmed also proceeding towards the P5 position, suggesting that different proteases may contribute to the proteolytic events of VH between RD and Ctrl.\u003c/p\u003e \u003cp\u003eMining of proteolytic events was then implemented with the search of C-termini by applying again a peptide-level FDR\u0026thinsp;\u0026le;\u0026thinsp;0.01. In this case, the main criteria for identification were the absence of a K or R residue at the C-terminus of the peptide.\u003c/p\u003e \u003cp\u003eMining of C-termini further expanded coverage of the VH degradome. In total, 149 C-termini were found as common to both the experimental groups, 46 exclusive of RD and 130 exclusive of Ctrl (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Whilst no significantly upregulated or downregulated C-termini were identified, in the case of Ctrl VH, C-termini for several crystallin proteins were detected, including a previously characterized MMP9 cleavage site within crystallin αB which is complementary to one of the neo N-termini identified for the same protein and discussed above. The full list of C-termini identified is provided in Supplementary Table\u0026nbsp;6.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows a schematic representation of biologically relevant N-termini and C-termini cleavages sites within the domain of proteins identified.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot validation of protein identification and proteolytic processing of osteopontin and vitronectin\u003c/h2\u003e \u003cp\u003eProteolytic processing of proteins was validated with manageable electrophoretic features (for this reason perlecan was excluded from analysis) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor each sample, 5 \u0026micro;g of VH proteins were separated and probed with antibodies raised against osteopontin (OPN), vitronectin (VTN), interphotoreceptor matrix proteoglycan 1 and 2 (IMPG-1 and IMPG-2, respectively) and DKK3. Given the semi-quantitative nature of the methodological approach and, at least in our opinion, the lack of a valid internal control for normalization purposes, we prefer not to speculate on quantitative data between groups.\u003c/p\u003e \u003cp\u003eIn the case of osteopontin, the immunostaining highlighted the typical pattern of the full-length protein (i.e., \u0026gt;\u0026thinsp;2 bands). Although an apparent increase of full-length osteopontin intensity in RD VH was observed, a long-exposure of filters highlighted the presence of a faint\u0026thinsp;~\u0026thinsp;30 kDa band only in the Ctrl group. This species was previously reported to correspond to the fragment released by MMPs degradation of the full-length protein\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, which also corresponded in MW to the cleavage site identified by mining of N-termini. Thus, Western blot analysis validated our proteomic and N-termini mining workflow.\u003c/p\u003e \u003cp\u003eIn the case of vitronectin, the full-length protein again showed a slight apparent increase in the RD VH group. However, in this case, several putative fragments were immunostained by the antibody over lower molecular weight regions of the filter exclusively in the RD group as well.\u003c/p\u003e \u003cp\u003eIn the case of IMPG1 and IMPG2 the pattern observed was unequivocal. The two full-length proteoglycans were in facts detected exclusively in RD samples. However, in the case of IMPG1 an additional band of approximately 50 kDa was identified across all samples. In the case of IMPG2 several immunoreactive bands were documented at MW lower than that of the full-fragments generated by cleavage of the glycoproteins within the sperm protein, enterokinase and agrin (SEA) domain could not be immunostained nor in Ctrl or RD samples, a putative fragment (\u0026lt;\u0026thinsp;45 kDa) with intensity comparable across all samples was documented for IMPG1 and diffuse fragments with unclear identity were stained for IMPG2.\u003c/p\u003e \u003cp\u003eWith regard to DKK3, whilst the band intensity showed comparable between samples, different potential cleavage patterns were observed especially for Ctrl VH samples.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this proteomic study, we coupled the characterization of VH proteome by LFQ DDA approaches with mining of natural N-termini (and C-termini), to introduce novel perspectives on the pathobiology of RD based on the identification of global perturbations of the proteome and of endogenous proteolytic events.\u003c/p\u003e \u003cp\u003eOur global analysis of the VH proteome was performed on a single subject scale (rather than pooled samples) and without immunodepleting for the most contaminating proteins. Despite the technical challenges associated with analysis of lower protein quantities of higher dynamic range, we were able to identify a robust number of proteins that was consistent with the existing literature, confirming key findings and further introduced novel observations\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Previous studies on RD and, in particular, PVR, agreed on a multifaceted dysregulation of energetic metabolism, redox unbalance, immune system regulation and growth factors bioavailability\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur study identified numerous dysregulated proteins and functionally enriched terms, with a sharp distinction between RD and Ctrl VH proteome.\u003c/p\u003e \u003cp\u003eSeveral proteins involved in TGF signaling, which is the main pro-fibrotic cytokine, or in pro-inflammatory cascades, such as TNF receptors, were also identified as markedly enriched in the RD VH proteome.\u003c/p\u003e \u003cp\u003eInterestingly, the polarization of macrophages towards the M2 phenotype has already been proposed as a driver of fibrosis and a major stimulus of RPE EMT during PVR; molecular cascades were effectively linked to TGFβ signaling\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA deeper examination of terms of CC, MF and BP processes, generated by submitting proteins upregulated and exclusive in RD VH to GO, highlighted further mechanisms that are very likely to underscore RD pathogenesis:\u003c/p\u003e \u003cp\u003eA wide repertoire of terms describing mechanisms of turnover and remodeling of the extracellular matrix, with particular emphasis on the photoreceptor outer segment (interphotoreceptor matrix proteoglycans), structural proteins (collagen, fibronectin) and receptors involved in cell adhesiveness and motility (e.g., selectins) were documented significantly enriched. In addition, mechanisms or regulation of peptidase and protease activity were observed, this being in accordance with previous findings, indeed\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and posing a rationale for subsequent exploration of endogenous proteolytic events.\u003c/p\u003e \u003cp\u003eIn addition to yielding insights into the global VH proteome, our dataset also allowed us to mine evidence of post-translational modification of VH proteins by proteolytic processing.\u003c/p\u003e \u003cp\u003eNatural endogenous N- and C-termini represent a minor fraction (~\u0026thinsp;5%) of identifiable peptides within a proteome. Prior to the relatively recent release of ultrafast search algorithms such as MSFragger, a good coverage of these peptides was only achieved by adopting enrichment strategies, such as N-tails\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Although this enrichment procedure would almost certainly boost the numbers of identified N-termini, the informatic pipeline adopted by us is recognized to retrieve a robust coverage of the N-terminome\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Effectively, we here identified and quantified\u0026thinsp;\u0026gt;\u0026thinsp;500 potential N-termini and \u0026gt;\u0026thinsp;300 C-termini. In most cases, they represent neo N-termini of structural and non-structural components of the VH that have not been reported before likely because the endogenous proteolysis of this fluid is an unexplored topic. However, several known cleavage sites were also identified. All together these data point out to specific events which may characterize the patho-physiological remodeling of the vitreous fluid.\u003c/p\u003e \u003cp\u003eTo test for specific enrichment of one or more proteases, data were submitted to TopFINDER (v. 4.1 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://topfind.clip.msl.ubc.ca/\u003c/span\u003e\u003cspan address=\"https://topfind.clip.msl.ubc.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e25\u003c/sup\u003e, a software specifically developed to characterize proteolytic events. This search retrieved evidence of the proteolytic activities of MMP9, CATD, CATS, MMP3 and MMP7 in Ctrl VH. A much broader array of enzymatic activities, including MMP12, MMP3, legumain, MEP1B, BMP1, were documented in RD VH. Although none of these proteases reached the statistical threshold (q\u0026thinsp;\u0026le;\u0026thinsp;0.05) for being considered dominant, the possibility of unbalanced proteolysis in RD VH, in our opinion, is robustly supported by four unrelated findings, namely: i) abundant VH proteins, such as crystallins, albumin and serotransferrin show different trends. Whilst neo N-termini of crystallins were significantly enriched in Ctrl VH, this being consistent with the tryptic datasets, neo N-termini of albumin and serotransferrin were more represented in RD VH in the absence of any obvious protein accumulation, at least based on inspection of the tryptic dataset; ii) the distribution of cleavage preferences between RD and Ctrl samples. Hydrophobic residues ranked first across P5 to P1\u0026rsquo; position of neo N-termini of Ctrl VH, whereas polar residues were mostly represented over the same sequence in the case of RD VH neo N-termini; iii) several mature and neo N-termini of natural inhibitors of proteases, such as α1-protease inhibitor, α1-antichymotrypsin, were identified in RD VH; iv) the pattern of neo N-termini of structural and non-structural elements was fairly different between the two experimental groups.\u003c/p\u003e \u003cp\u003eWith respect to this last point, the total repertoire of N-termini (including RD and Ctrl) suggests that proteolysis may in particular affect mechanisms of immune system regulation and cell adhesiveness/angiogenesis.\u003c/p\u003e \u003cp\u003eRegarding the immune system, it is worth pointing out that mature and neo-termini, either C- or N-, were identified for complement components together with a previously identified neo N-terminus of natural killer cell-enhancing factor B in RD VH samples. Considering also the findings retrieved by the tryptic searches discussed above, it is likely that dysregulation of immune system polarization progressively develops in RD subjects.\u003c/p\u003e \u003cp\u003eDiscussion of proteolysis-mediated mechanisms of angiogenesis regulation, instead, is enriched with several interesting findings.\u003c/p\u003e \u003cp\u003eA neo N-termini stretching across the 186\u0026ndash;193 residues of osteopontin (assigned to isoform 4 by FragPipe but shared with wild type osteopotin) and previously described to be released by a MMP3/MMP7 cut at \u003csup\u003e185\u003c/sup\u003e\u0026darr;Ala\u003csup\u003e186\u003c/sup\u003e was identified in Ctrl VH. The cleavage of osteopontin was reinforced by the identification of a further C-terminus of the protein (in both Ctrl and RD VH) and by the Wb approach, which highlighted the presence of a faint 30 kDa fragment, but compatible with that released by MMP3 and MMP7\u003csup\u003e22\u003c/sup\u003e. Osteopontin is a sialoprotein serving key roles for the physiological composition of extracellular matrix\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Importantly, the fragment here detected was reported to have bioactive properties for cell adhesiveness, migration and angiogenesis.\u003c/p\u003e \u003cp\u003eRemarkably, based on search on tryptic peptides, osteopontin was documented as robustly upregulated in the RD VH, suggesting that these subjects may experience an increase in protein level through reduced proteolysis. In a previous study, protein levels were strongly increased in the VH of PVR subjects, compared to subjects without this complication. This finding stimulated the authors to associate osteopontin levels to RD, probably through impaired angiogenesis \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn coherence with this scenario, two major structural glycoproteins, such as basement membrane-specific heparan sulfate proteoglycan core protein, also called perlecan, and vitronectin were identified with neo N-termini in RD VH.\u003c/p\u003e \u003cp\u003ePerlecan, is a major component of the inner limiting membrane (ILM) and VH body. Correct synthesis and deposition of this glycoprotein, as well as its turnover, are key for the homeostasis of these two ECM components of the eye\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In our study, a perlecan neo N-terminus (4196\u0026ndash;4222 residues) was found to stretch across the C-terminal fragment of the protein. This fragment, called endorepellin, is released by BMP1 cleavage at \u003csup\u003e4195\u003c/sup\u003e\u0026darr;Glu\u003csup\u003e4196\u003c/sup\u003e and was reported to have strong angiostatic activities by inhibiting endothelial cell adhesion to fibronectin and type I collagen\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVitronectin, instead, is a glycoprotein synthesized and released by photoreceptors, but also a component of plasma \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e which play pivotal roles in ECM stabilization as well as regulation of vessels genesis and sprouting across the matrix layers. Vitronectin fragments were identified as neo N- and C-termini in RD VH and a possible fragmentation pattern of the protein was observed also by Wb, although, the abundance of the full length protein, by this approach, was probably higher in RD vs Ctrl VH. Nevertheless, dysregulated vitronectin levels were documented by a proteomic characterization of the VH isolated from patients with blood veins occlusions, confirming that even in the retina, the glycoprotein is expected to serve roles in vessels microarchitecture and, thereby blood hemodynamics. As widely discussed throughout the manuscript, these are both patho-physiological processes considered altered in RD and PVR subjects \u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to these main findings, a proteolysis-based dysregulation of angiogenesis is supported by additional evidence, including detection of neo N-terminus of cystatin 3, which promotes angiogenesis, and of PEDF (serpin F1), in RD VH.\u003c/p\u003e \u003cp\u003eAmong N-termini identified, it is worth commenting the case of IMPG1 and IMPG2. These are (both) secreted and membrane glycoproteins that serve key role for the physiological composition and homeostasis of the interphotoreceptor matrix which surround the inner and outer segment of photoreceptors. Interestingly, the biological activity of these glycoproteins is dependent on the proteolytic activation of the immature protein by an enzymatic cut within the SEA domain. Mutations in the SEA domain that impair proteolytic processing of IMPG1 and IMPG2 are associated with retinitis pigmentosa (RP)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, IMPG1 and IMPG2 were documented as robustly upregulated (Log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026gt;\u0026thinsp;3, p.ord\u0026thinsp;\u0026le;\u0026thinsp;0.05) in RD VH, compared to Ctrl VH, when the search was run using the tryptic dataset, confirming previous studies\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and exclusive of RD VH, at least for the sensitivity of the approach, by Wb. By deeply examining the PSMs, it is remarkable that, in the case of IMPG2, 12 high confident (FDR\u0026thinsp;\u0026le;\u0026thinsp;0.01) tryptic peptides were observed in RD VH, and only 3 in Ctrl VH.\u003c/p\u003e \u003cp\u003eHowever, in the case of IMPG2, a neo N-terminus was instead identified as robustly downregulated in RD VH (Log\u003csub\u003e2\u003c/sub\u003eFC = -0.734, p.ord\u0026thinsp;\u0026lt;\u0026thinsp;0.444), whilst a neo C-terminus of the same protein was exclusively identified in Ctrl VH. The discrepancy between the tryptic and neo termini datasets envisages the possibility that accumulation of IMPG2 in RD VH, which is likely caused by photoreceptor degeneration, is not accompanied by their proteolytic processing, which is, instead, key for their biological function.\u003c/p\u003e \u003cp\u003eThis study has some limitations to acknowledge. Pitfalls of present study include the relatively small sample size and the absence of longitudinal data limiting commentary on their link to disease progression, in particular PVR. Larger well-controlled studies are required to confirm these pilot findings.\u003c/p\u003e \u003cp\u003eAn additional hypothesis that demands careful attention for the interpretation of this set of data and the additional ones that will be generated by forthcoming studies is that some of the proteins observed may display divergent compartmentalization within the same fluid. This is the case of extracellular vesicles (e.g., exosomes), which are released as a consequence of the degeneration the photoreceptors partially undergo during the acute phase of RD\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Sequestration of a protein inside a vesicle shields it from digestion and identification.\u003c/p\u003e \u003cp\u003eFurthermore, many of the proteins here identified are heavily glycosylated under physiological conditions. Glycosylation is a PTM which critically protects them from proteolytic digestion in vivo and in vitro. Therefore, to obtain a thorough picture the glycosylation patterns should be investigated too.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn conclusion, our findings introduce the working hypothesis that RD VH is characterized by altered proteolysis of structural and non-structural components. Some of the findings here reported, in particular those supporting a proteolysis-based alteration of angiogenic processes may represent a molecular rationale for the morphological and functional abnormalities of vessels microarchitecture RD subjects have been reported to develop. Conceptually, it is very difficult to speculate whether the alterations observed contribute to RD onset or are a consequence of the disease. In this last case, it may be relevant to investigate whether they predispose to recurrent RD and PVR.\u003c/p\u003e \u003cp\u003eTherefore, further studies on a larger cohort of samples and using additional enrichment strategies are demanded to clarify the pathogenic role of the proteolytic alterations here identified. We nonetheless believe that the approach here undertaken has the merit to pose a scientific question that may foster novel pathogenetic perspectives on RD pathobiology and, hopefully, new therapeutic targets and potential biomarkers to predict the recurrence of RD and/or PVR.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAqueous Humor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBicinchoninic acid assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBiological Process\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCART\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClassification and regression trees\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCellular Component\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eData Dependent Acquisition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferentially Expressed Protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDTT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDithiothreitol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial to Mesenchymal Transformation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eILM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInner Limiting Membrane\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLFQ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLabel Free Quantification\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMolecular Function\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOPN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOsteopontin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrincipal Component Analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProteome Discoverer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePars Plana Vitrectomy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePTM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePost-Translational Modification\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePVR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProliferative Vitreo-Retinopathy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRetinal Detachment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRetinitis Pigmentosa\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRPE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRetinal Pigment Epithelium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRRD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRecurrent RD\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSperm protein, Enterokinase and Agrin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTrifluoroacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUb\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUbiquitin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUltra High Performance Liquid Chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVitreous Humor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVTN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVitronectin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare that do not have pending conflict of interest with the study reported\u003c/p\u003e \u003ch2\u003eSUPPORTING DATA\u003c/h2\u003e \u003cp\u003eData presented in this manuscript are supported by additional supporting information. Supplementary Figure and Table files have been uploaded.\u003c/p\u003e \u003ch2\u003eFUNDING INFORMATION\u003c/h2\u003e \u003cp\u003eThe authors further received financial support from LazioInnova (grant: A0375-2020-36591). This study was supported by Next Generation Promising (NGP) and PRIN-MIUR (grant: 2022R9WCZS_001).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eG.A.Z.: conceptualization, data curation, formal analysis, investigation, methodology, software, writing; S.G.: conceptualization, data curation, formal analysis, software, writing; I.P.: data curation, formal analysis; L.P., G.R., G.R.T., G.G., A.B.: conceptualization, formal analysis, supervision and validation; P.A.B.: conceptualization, data curation, investigation, methodology; T.R.: writing \u0026ndash; review and editing, supervision, resources; D.S.: conceptualization, data curation, investigation, writing \u0026ndash; review and editing, supervision, resources.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge the Ministry of Health and Fondazione Roma for the support. G.A. Zingale and I. Pandino were supported by the PhD program in Chemical Sciences, University of Catania.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003e\"The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD057155 and 10.6019/PXD057155\" (login details for reviewing process, Username: [email protected]; Password: 4ZnKepzd6UaA)37.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSultan, Z. N., Agorogiannis, E. I., Iannetta, D., Steel, D. \u0026amp; Sandinha, T. Rhegmatogenous retinal detachment: a review of current practice in diagnosis and management. \u003cem\u003eBMJ Open. Ophthalmol.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e, e000474 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIdrees, S., Sridhar, J. \u0026amp; Kuriyan, A. E. Proliferative Vitreoretinopathy: A Review. \u003cem\u003eInt. Ophthalmol. Clin.\u003c/em\u003e \u003cb\u003e59\u003c/b\u003e, 221\u0026ndash;240 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMomenaei, B. et al. Incidence and Outcomes of Recurrent Retinal Detachment after Cataract Surgery in Eyes with Prior Retinal Detachment Repair. \u003cem\u003eOphthalmol. Retina\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e, 447\u0026ndash;455 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGe, J. Y. et al. International incidence and temporal trends for rhegmatogenous retinal detachment: A systematic review and meta-analysis. \u003cem\u003eSurv. Ophthalmol.\u003c/em\u003e \u003cb\u003e69\u003c/b\u003e, 330\u0026ndash;336 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitry, D., Charteris, D. G., Fleck, B. W., Campbell, H. \u0026amp; Singh, J. The epidemiology of rhegmatogenous retinal detachment: geographical variation and clinical associations. \u003cem\u003eBr. J. Ophthalmol.\u003c/em\u003e \u003cb\u003e94\u003c/b\u003e, 678\u0026ndash;684 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMudhar, H. S. A brief review of the histopathology of proliferative vitreoretinopathy (PVR). \u003cem\u003eEye (Lond)\u003c/em\u003e. \u003cb\u003e34\u003c/b\u003e, 246\u0026ndash;250 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, W. X., Xing, M. \u0026amp; Apte, R. S. Interventions for Proliferative Vitreoretinopathy. \u003cem\u003eJAMA Ophthalmol.\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1001/jamaophthalmol.2024.1093\u003c/span\u003e\u003cspan address=\"10.1001/jamaophthalmol.2024.1093\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrispin, M. et al. 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The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, D543\u0026ndash;D552 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Vitreous humor, proteomics, N-termini, proteolysis, angiogenesis","lastPublishedDoi":"10.21203/rs.3.rs-5395071/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5395071/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRetinal detachment is a severe eye condition characterized by the detachment of the neurosensory retina from the retinal pigment epithelium and caused by retinal tears.\u003c/p\u003e \u003cp\u003ePars plana vitrectomy is the elective surgical procedure during which vitreous humor is collected. This fluid shapes the eye globe providing mechanical and nutritional support to the retina.\u003c/p\u003e \u003cp\u003eHence, exploring the proteome of vitreous humor isolated from subjects diagnosed with retinal detachment is supposed to help decipher the pathobiology of the disease and that of its complications, such as proliferative vitreo-retinopathy, which predispose to recurrent RD (observed in 20% of cases), a sight threatening condition.\u003c/p\u003e \u003cp\u003eHerein, we investigated the perturbations of vitreous proteome between subjects affected by primary retinal detachment and controls by shot-gun proteomics approaches.\u003c/p\u003e \u003cp\u003eSpectra were first searched and analyzed to identify proteome perturbations. Thereafter, starting from the hypothesis that the disease could be sustained by altered proteolytic processing of structural and non-structural elements of vitreous humor, N- and C-termini were mined to uncover endogenous proteolytic events.\u003c/p\u003e \u003cp\u003eThis search retrieved evidence of a wide repertoire of proteolytic events and proteolytic sites, either already described for proteins commonly identified also in other biological samples, or likely specific of this fluid. Comparison between the N- and C-termini landscapes and the perturbations of global proteome highlighted robust alterations of the repertoire of cleaved proteins between retinal detachment and control subjects. Strengthened by immunoblotting studies on a selection of proteins, datasets envisage that retinal detachment is characterized by unbalanced proteolysis of structural and non-structural components involved in the regulation of immune processes, proteolytic control and, in particular, angiogenesis.\u003c/p\u003e","manuscriptTitle":"Altered Pattern of Proteolysis of Extracellular Proteins in Rhegmatogenous Retinal Detachment by Mining of Tryptic and N-Termini Datasets From Vitreous Humor Proteome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-02 22:59:52","doi":"10.21203/rs.3.rs-5395071/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-23T12:51:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-26T10:10:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299471259747091334834264598724399005380","date":"2024-11-12T20:28:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"97053602144001660742914700318098438058","date":"2024-11-11T11:48:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-11T11:24:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-11T11:22:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-08T02:58:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-05T12:57:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-11-05T11:27:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5c8b3907-6d29-41bc-a7df-97cb725cf84a","owner":[],"postedDate":"December 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40469020,"name":"Biological sciences/Biochemistry"},{"id":40469021,"name":"Biological sciences/Biochemistry/Proteases"},{"id":40469022,"name":"Biological sciences/Biochemistry/Proteolysis"},{"id":40469023,"name":"Biological sciences/Biochemistry/Proteomics"},{"id":40469024,"name":"Health sciences/Biomarkers/Predictive markers"}],"tags":[],"updatedAt":"2025-10-20T16:01:08+00:00","versionOfRecord":{"articleIdentity":"rs-5395071","link":"https://doi.org/10.1038/s41598-025-19857-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-14 15:57:27","publishedOnDateReadable":"October 14th, 2025"},"versionCreatedAt":"2024-12-02 22:59:52","video":"","vorDoi":"10.1038/s41598-025-19857-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-19857-z","workflowStages":[]},"version":"v1","identity":"rs-5395071","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5395071","identity":"rs-5395071","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}