{"paper_id":"20da36fa-4a44-4165-b113-2b5606a4e97c","body_text":"1 \nA journey towards developing a new cleavable crosslinker reagent \nfor in-cell crosslinking \n \nFränze Müller 1*☼, Bogdan Razvan Brutiu 2*, Iakovos Saridakis 2, Thomas Leischner2, Micha \nBirklbauer3, Manuel Matzinger 1, Mathias Madalinski1, Thomas Lendl 1, Saad Shaaban2, \nViktoria Dorfer3◊, Nuno Maulide2◊, Karl Mechtler1◊ \n \n1Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria.  \n2Institute of Organic Chemistry, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria.  \n3Bioinformatics Research Group, University of Applied Sciences Upper Austria, 4232 Hagenberg, \nAustria. \n \n☼Submitting author \n*Contributed equally, shared first author \n◊Corresponding authors: E-mail(s): karl.mechtler@imp.ac.at; nuno.maulide@univie.ac.at; \nviktoria.dorfer@fh-hagenberg.at  \n \nKeywords: crosslinking mass spectrometry, click reaction, in -cell crosslinking, enrichment,  \nchemical coupling, fragmentation behaviour \n \nAuthor contributions: \nFränze Müller  conceptualized the study, designed the MS and microscopy -based \nexperiments, performed data analysis, wrote the manuscript (MS based & microscopy based), \ndesigned the figures; Bogdan R. Brutiu synthesised the DiSPASO cross-linker and wrote the \nchemistry part of the manuscript, Iakovos Saridakis wrote the chemistry part of the FWF \nproposal and designed the cross -linker, Thomas Leischner synthesised the picolyl azide  , \nMicha Birklbauer  adapted MS Annika for additional doublet searches and wrote t he \ncorresponding method section; Manuel Matzinger conceptualized and wrote the main part of \nthe FWF proposal and provided data for DSBSO analysis; Matthias Madalinkski synthesised \nthe peptide used in this study and wrote the corresponding method section; Thomas Lendl \nquantified the fluorescence signals of microscopy experiments and wrote the corresponding \nmethod section; Saad Shaaban  coordinated the chemical experiments and wrote the \nchemistry part of the manuscript; Karl Mechtler , Nuno Maulide  and Viktoria Dorfer  \nsupervised the study. All authors revised and agreed on the manuscript. \n \nGraphical Abstract \n \n \n \n \n \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n2 \nAbstract \nCross-linking mass spectrometry (XL-MS) is a powerful technology that recently emerged as \nan essential complementary tool for elucidating protein structures and mapping interactions \nwithin a protein network. Crosslinkers which are amenable to post-linking backbone cleavage \nsimplify peptide identification, aid in 3D structure determination and enable system -wide \nstudies of protein-protein interactions (PPIs) in cellular environments. However, state -of-the-\nart cleavable linkers are fraught with practical limi tations, including incomplete and harsh \nfragmentation operations. \nWe herein introduce DiSPASO as a lysine-selective, MS-cleavable cross-linker with an alkynyl \nhandle for affinity enrichment. DiSPASO was designed and developed for efficient cell \nmembrane permeability and cross-linking while securing low cellular perturbation. We tested \nDiSPASO employing three different copper-based enrichment strategies using model systems \nwith increasing complexity (Cas9 -Halo, purified ribosomes, live cells). Fluorescence \nmicroscopy in-cell cross-linking experiments revealed a rapid uptak e of DiSPASO into HEK \n293 cells within 5 minutes. While DiSPASO represents progress in cellular PPI analysis, its \nlimitations require a careful strategy, highlighting the complexity of developing effective XL -\nMS tools and the importance of continuous innov ation in accurately mapping PPI networks \nwithin dynamic cellular environments. \n \nIntroduction \nOver the past decades, cross -linking mass spectrometry (XL -MS) has developed as a \npowerful complementary technique for studying the spatial arrangement of proteins and their \ninteractions within complexes. MS-cleavable cleavable cross-linkers, a class of cross-linkers \namenable to backbone fragmentation upon MS acquisition, have gained attention in XL -MS \nby greatly simplifying the MS analysis and facilitating the identification of crosslinked peptides. \nThe data analysis process is supported by the generation of characteristic doublets of \nfragment ions during MS2 fragmentation, eventually enabling straightforward and accurate \nidentification of cross -links even in complex mixtures, such as cell lysates, whole cells or \ntissues. Consequently, cleavable cross-linkers have significantly enhanced structural analysis \nby offering precise distance constraints, intrinsic to their chemical properties. This capability \nis particularly valuable for resolving the three-dimensional architecture of proteins and protein \ncomplexes, especially when combined with complementary techniques such as HDX -MS or \ncryo-EM1–7. Moreover, cleavable crosslinkers have enabled system -wide XL -MS studies, \ncapturing protein -protein interactions (PPIs) in cellular environments, including weak or \ntransient interactions 8–11. This advancement allows for the exploration of the structural \ndynamics of proteins in their native state. Despite advancements in data analysis, there remain \nchallenges in accurately determining false discovery rates (FDR) for identified PPIs. \nFurthermore, the complexity of the data requires careful handling of error estimation to ensure \nthe reliability of reported PPIs12.  \nAdditionally, there are practical limitations such as the requirement for specific conditions for \ncleavage, the potential for incomplete cleavage, and the need for specialized mass \nspectrometry setups to detect and analyze cleavage products effectively 13. Hence, \ncrosslinkers are often combined with enrichable groups, such as biotin14–18, known for its high \naffinity towards streptavidin or avidin, and sometimes with MS-cleavable groups, for proteome-\nwide analyses 19–21. However, the strong streptavidin -biotin interaction can raise difficulties \nupon release from the solid support, prompting alternative strategies like solid -supported \nmonomeric avidin or incorporating release groups in the crosslinker backbone 17,22,23. Various \nrelease groups, including PEG 14, pinacol esters 14,19, azobenzenes 16, disulfides 16,24, and \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n3 \nphotocleavable groups16, have been explored. Click chemistry groups offer versatility, serving \nas capture or enrichment groups, with reversible options like acid-cleavable acetal or disulfide \nbonds24–30. While this innovation marks significant progress, it also presents opportunities for \nfurther refinement, as demonstrated by the development of a negatively charged phosphonic \nacid as a crosslinker reagent 31. While this offers simplicity, its negative charge inhibits cell \nmembrane crossing, hindering in -cell crosslinking. This hurdle could be overcome by \nprotecting the negative charge with a chemical protection group 32. Chemical modifications in \ncrosslinking reagents offer promising functionalities but also pose challenges like increased \nhydrophobicity. Cleavable crosslinkers have advanced XL -MS, enhancing protein structure \nanalysis, though further improvements are needed to overcome current limitations.  \nTo overcome the aforementioned limitations, we hereby introduce DiSPASO ( 1) as a novel \nlysine reactive, MS -cleavable and membrane -permeable crosslinker featuring an alkene -\nbased click chemistry handle for affinity enrichment. This study provides a detailed exploration \nof the design, synthesis, and careful characterization of DiSPASO, providing a comprehensive \nevaluation of its chemical properties, effectiveness in mapping protein -protein interactions, \nand performance relative to existing crosslinkers. This analysis will also explore DiSPASO's \nfragmentation behaviour in mass spectrometry, highlighting both its advantages and the \nchallenges encountered, thereby offering insights into its potential and areas for further \noptimization in crosslinking mass spectrometry applications. \n \nResults \n \nSynthesis of a novel cleavable and enrichable crosslinker reagent DiSPASO and its modular \nperspective \nWe designed the crosslinker DiSPASO ( 1) bearing 4 key elements: a stable underexplored \ncore decorated with an enrichment site ( terminal alkyne), two side -chains containing the \ncleavable sulfoxide moieties and terminal NHS -esters for targeting lysins. A retrosynthetic \nanalysis of compound (1) suggested a common intermediate S5 in the synthesis pathway. S5 \nwas efficiently synthesized from commercially available diester amine S1 through a series of \nreactions: a Sandmeyer reaction to introduce the aryl bromide, reduction of the ester groups, \nan Appel-type bromination and finally substitution with the corresponding methyl ester-bearing \nthiol. S5 was obtained on a gram scale. The aryl bromide moiety presents an ideal candidate \nfor cross -coupling with various enrichment sites. In this study, we chose a Sonogashira \ncoupling. A global deprotection to the diacid, followed up by NHS coupling formed pre cursor \nS8a. Optimized oxidation delivered the novel enrichable cross-linker DiSPASO (1) in over 150 \nmg ( Figure 1A ). With  a streamlined synthesis in hand, the NHP derivative ( 2) was also \nobtained after two simple steps (Figure 1B). The high-yielding steps can easily be scaled up. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n4 \n \nFigure 1: (A) Design and synthesis of DiSPASO. (B) Synthetic route: straightforward synthesis of \nDiPPASO. aSandmeyer Reaction: 1.2 eq. NaNO 2/HBr, 0 °C, 1h, 1.4 eq. CuBr, 0 °C, 18 h. bReduction: \n2.0 eq. LiAlH 4, THF, 0 °C, 2 h. cBromination: 8.5 eq. PBr 3, DCM, 40 °C, 15 h, then H 2O, 23 °C, 20 h. \ndSubstitution: 2.05 eq. methyl 3 ‑mercaptopropanoate, 2.05 eq. K 2CO3, DMF, 40 °C, 30 h. eCross-\ncoupling: 1.0 eq. TMS‑acetylene, 0.05 eq. Pd(dppf)Cl2•CH2Cl2, 0.05 eq. CuI, THF, 60 °C, 20 h. fGlobal \ndeprotection: 6.0 eq. LiOH, THF/H2O (3:1), 23 °C, 20 h. gNHS/NHP coupling: 2.2 eq. NHS or NHP, 2.2 \neq. EDCI•HCl, 0.2 eq. NEt3, DMF, 23 °C, 24 h. hOxidation: 2.0 eq. mCPBA, DCM, 0 °C, 2 h. \n \nDiSPASO in context to other in-cell and lysate crosslinkers \nDiSPASO was engineered as an in -cell crosslinker reagent with good cell membrane \npermeability properties while maintaining the solubility of the reagent during the in -cell \ncrosslinking procedure. For a direct comparison of DiSPASO in the context of other s tate-of-\nthe-art crosslinkers used in in -cell or cell lysate crosslinking, we used a topological polar \nsurface area (tPSA) against partition coefficient (cLogP) plot ( Figure 2). This visuali zation \noffers critical insights into the chemical properties of com pounds, essential for design and \noptimization purposes. It effectively showcases the delicate balance between hydrophilicity \nand lipophilicity, crucial for understanding a compound's permeability across biological \nmembranes. Compounds exhibiting lower tPSA  values and higher cLogP values tend to \ndisplay enhanced membrane permeability, owing to their greater affinity for lipid bilayers. \nDiSPASO shows medium membrane permeability and hydrophobicity properties compared to \nother crosslinker reagents and is in line with the BSP crosslinker published in Gao et al.25,26. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n5 \nTrifunctional crosslinkers, such as DiSPASO, enhance crosslinking mass spectrometry \ninvestigations by boosting sensitivity and specificity, primarily through an affinity tag that aids \nin enriching low-abundant crosslinked peptides that are typically masked  by abundant linear \npeptides. This advancement is crucial for uncovering elusive protein interactions and enriching \nXL-MS data depth. DiSPASO's effectiveness in cellular entry and membrane permeation was \nevaluated using three different enrichment strategie s in increasing sample complexities \nstarting from single peptide crosslinking towards live HEK cell crosslinking. \n \n \nFigure 2: Topological Polar Surface area plot of commonly used crosslinker reagents. DiSPASO is \nshown in the center of the plot (green dot) with medium membrane permeability and hydrophobicity \ncompared to other common crosslinker regents. DiPPASO (not presented in this study) shows similar \nproperties to t -BuPhoX (TBDSPP) with a high hydrophobicity compared to other cro sslinkers. The \nclouds of enrichable and permeable crosslinkers are clustered in two distinct parts of the plot, with \nDiSPASO at the interface  between both groups. Classification of the crosslinkers was extracted from \nthe Thermo Scientific crosslinker selection tool. If a crosslinker shoes two properties of the \nclassifications the more dominant property was selected for plotting.  \n \nComparison of DSBSO and DiSPASO crosslinking using Cas9-Halo   \nWe optimized the affinity enrichment by assessing the following strategies (Figure 3). The first \nenrichment employing picolyl azide (Figure 2A, (3)) as a click handle and immobilized metal \naffinity chromatography (IMAC) for crosslinked peptide enrichment was tested using a single \nsynthetic peptide (Ac-WGGGGRKSSAAR-COOH) with a defined crosslink-site (Figure S1A) \nand additionally using Cas9 -Halo as the model protein ( Figure S2A ) to ensure a fully \ncontrolled environment. Both experiments demonstrated maximal crosslinked \npeptides/intensity when using 5  mM picolyl azide, with a click reaction efficiency of 99.3%. \nClick reaction efficiency was monitored by converting crosslinked products from DiSPASO \ncrosslinking (Figure S2A, blue bars) to products formed after click reaction with picolyl azide \n(Figure S2A, orange bars), completing the click reaction when all DiSPASO links converted \nto click product BPNP (4) in Figure 3A. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n6 \n \nFigure 3: Overview of DiSPASO enrichment strategies. A: DiSPASO enrichment using a picolyl azide \nas a click chemistry reagent and an IMAC -based enrichment strategy to retrieve crosslinked peptides \nfrom a complex mixture. B: Azide-S-S-biotin (ASSB) based enrichment using ASSB as a click chemistry \nreagent and biotin -streptavidin bead strategy to enrich for crosslinked peptides. The elution of bead -\nbound crosslinked peptides is performed using the reduction of the disulfide bond of the ASSB \ncompound. C: Simplified version of B. The click reagent Disulfide Azide is already coupled to the beads \nand the click reaction is taking place directly on the beads. Elution of crosslinked peptides is performed \nafter the click reaction and washing procedure of the beads using a reducing reagent. IUPAC names of \nall compounds used in this manuscript are described in Table S4. \n \nThis principle was applied to all three enrichment strategies, tracking efficiency by monitoring \nmass shifts after each reaction's conversion. Following click reaction optimization, the optimal \nhigher-energy collision dissociation (HCD) was determined via single peptide crosslinking. \nThe best-combined score for a crosslinked peptide was achieved with an HCD of 34 (Figure \nS1B), despite the main doublet intensity decreasing with higher HCDs ( Figure S1C ). A \nbalance between peptide backbone fragmentation and doublet intensity required for crosslink \nidentification was essential. Consequently, optimal fragmentation energies of 25, 27, and 32 \nwere selected for further experiments. \nThe fragmentation pattern of DiSPASO was evaluated on Cas9 crosslinking data (Figure 4). \nSix potential fragments for the DiSPASO crosslinker were calculated based on the \nsubstitutions of the structures shown in Figure 4A . The full crosslinker mass was also \nconsidered as fragment 7 to account for the theoretical cleavage of NH -crosslinker/ peptide \nbounds. Fragment ETFP (9), ETHMP (10) and alkene (11) represent the common and \nexpected fragmentation pattern that is known fro m several fragmentation studies rega rding \nDSBSO19,33 and DSSO 10,34,35. Additionally, a second theoretical fragmentation pattern was \ntested due to low identification rates using DiSPASO for Cas9 crosslinking. The second \nfragmentation pattern includes a sulfenic acid fragment (13) or a thiol fragment (14) on the \nshort side an d an unexpected fragment EMP (12) on the long side of the crosslinker. Each \nfragment was defined separately according to the monoisotopic masses in Figure 4D&E. \nPossible doublet distances were calculated according to equation 1 in the method section \n“Software adjustments”. This resulted in theoretical 15 doublet distances that can occur during \nfragmentation of DiSPASO. The same procedure was applied to DSBSO to compare the \nfragmentation behaviour of both crosslinkers. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n7 \n \nFigure 4: Possible fragments of DiSPASO during MS2 fragmentation and evaluation of MS Annika \nsearch setting regarding doublet distances. A: Fragmentation products of DiSPASO after high -\nperformance collision-induced dissociation showing structures of DSSO-like cleavage products (alkene \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n8 \n11, ETFP 9 and ETHMP-fragments 10, main doublet) and possible additional doublets of the long side \nof the crosslinker (EMP 12 with SA 13 or T 14 as stubs, dotted grey box). B: Numbers of residue pairs \nidentified from Cas9 crosslinked with DiSPASO while searching with each doublet distance separately. \nD12 (ETHMP-alkene doublet) shows the maximum number of identified links with D11 (ETFP -alkene) \nas the second and D1, D2 (alkene -thiol, sulfenic acid -alkene) as the third abundant doublets. C: \nNumbers of residue  pairs identified from Cas9 crosslinked with DSBSO while searching with each \ndoublet distance separately. D11 (ETFP-alkene doublet) shows the maximum number of identified links. \nD: Table of doublet definitions and delta masses of all possible doublets. E: Table of substitutions and \nmonoisotopic masses of all fragments including DiSPASO as full construct bound to peptides. IUPAC \nnames of all compounds used in this manuscript are described in Table S4.  \n \nAzide-DSBSO shows similar chemistry on the crosslinker backbone since it has the same \nlength and cleavage site as DiSPASO. The cent er of Azide-DSBSO also comprises a ring \nsystem but in contrast to DiSPASO it is not a conjugated pi -ring system and instead of an \nalkene functionality DSBSO employs an azide group for the enrichment and click chemistry. \nHence, DSBSO was considered an ideal p ositive control to compare to the fragmentation \npattern that was already published before19,34.  \nSurprisingly, DiSPASO showed various fragmentation sites with doublet D11 and D12 as the \nmain occurring doublet distances. D11 represents the doublet distance between ETFP (9) and \nalkene (11) with a distance of 208 Da. The second main doublet is D12, ETHMP and alkene \nwith a distance of 226.01  Da. Both doublets belong to the known fragmentation pattern \npublished for DSSO before 34. Additionally, DiSPASO shows doublets for the less likely \nfragmentation pattern between fragment EMP (12) and sulfenic acid (13) o r thiol (14), D13 \nand D14 respectively, and alkene and thiol or sulfenic acid alone, D1 and D2 respectively. \nWhereas D13 and D14 represent 25% and 17% of the identified links, respectively, D1 and \nD2 account for 30% and 34%, highlighting the increased backbone fragmentation of the \nDiSPASO crosslinker. In contrast, DSBSO shows only one main doublet distance D11, which \nis defined by a distance of 182  Da between the alkene fragment and the long thiol fragment \nof DSBSO. D11 resulted in 285 identifiable links on average. D1 and D5 represent the second \nmost abundant doublet distances with 16% and 15%, respectively. Although DSBSO also \nshows additional doublet distances, the main doublet distance stands out compared to all \nothers. In conclusion, DiSPASO shows a different fragmentation pattern, that yields an almost \ncomplete scattering of the crosslinker backbone, compared to DSBSO, although the main \ncleavage sites are the same in perspective of the backbone. Hence, for further DiSPASO \nanalysis the main doublet distances D11 and D12 were employed for crosslink identifications \nin MS Annika.  \nWith all optimised parameters in place, the picolyl azide enrichment yielded 312 unique \nresidue pairs compared to 259 identified residue pairs in the non -enriched control sample \n(Figure 5A). The picolyl enrichment increased the number of links by 17% but the overall \nperformance compared to DSBSO is lowered by 33% for 500ng (695 links DSBSO, 465 links \nDiSPASO) and 29% for 200ng injection amount (363 links DSBSO, 259 links DiSPASO). \nNevertheless, the technical reproducibility of DiSPASO crosslinks before and after enrichment \nis high with 37% and 47% overlap between triplicates, respectively (Figure 5B). The recovery \nrate of crosslink before and after enrichment is 49% with 21% uniquely identified in the \nenriched sample.  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n9 \n \nFigure 5: Comparison of DiSPASO and DSBSO using Cas9 as a model protein, as well as enrichment \nperformance of DiSPASO using picolyl azide as enrichment tag. A: Number of identified unique \ncrosslinks (unique residue pairs) in a triplicate experiment of Cas9 crosslinked with either DiSPASO or \nDSBSO in different injection amounts (500 and 200 ng) Blue indicates non -enriched crosslinked \npeptides, yellow enriched crosslinked peptides from Cas9 DiSPASO experiments. The green colour \nrepresents Cas9 crosslinking results using DSBSO. Dots on top of the bar show individual residue pair \nnumbers of each replicate. The median value of residue pairs is plotted in the middle of the bar. B: \nOverlap of identified crosslinks after click reaction and enrichment using picolyl azide as click chemistry \nreagent. After click reaction and enrichment using picolyl azide the numbers of crosslinks show an \noverlap of 20%. Left bottom of panel B: Overlap of technical replicates of non-enriched Cas9 links. Right \nbottom of panel B: Overlap of technical replicates of enriched Cas9 links. \n \nThe picolyl azide enrichment strategy was tested in a more complex system with crosslinked \nCas9 as spike-in and HeLa cell lysate as background to increase the overall complexity of the \nsample ( Figure 6A, Figure S2B ). The spike -in amount ranged from 0.5  ug to 10  ug \ncrosslinked Cas9 in 100 ug HeLa lysate. The lower the spike -in amount the fewer crosslinks \ncould be identified after enrichment. For 0.5 ug only 14 crosslinks and the 1:100 ratio (near in-\ncell condition) 20 crosslinks of a triplicate injection could be ident ified. In-cell crosslinking \nexperiments in HeLa cells were performed to prove the hypothesis that the picolyl azide \nenrichment strategy is underperforming in real -case scenarios. Indeed, for HeLa in -cell \ncrosslinking only 1 crosslink could be identified ( Figure 6A, grey background). To improve \nthe enrichment of crosslinked peptides in in -cell crosslinking experiments we moved to a \ndifferent enrichment strategy using an Azide-S-S-biotin click chemistry handle.  \n \nAzide-S-S-biotin and Disulfide azide agarose bead enrichment  \nAzide-S-S-biotin (ASSB), known for its effectiveness in protein and peptide labelling and \nenrichment, was utilized to enhance crosslinked peptide enrichment in in -cell crosslinking, \nleveraging its cleavable disulfide bond for peptide release post -enrichment (Figure 3B). The \nstrong binding of the biotin group to streptavidin beads can be fully exhausted, while binding \nefficiency can be granted due to the reduction of the disulfide bond. The resulting enriched \nand cleaved crosslinked peptide is small in size, which ensures minimal impact on ionization \nefficiency during the electron spray ionization (ESI) process. \nASSB's optimization was conducted using the Cas9-Halo model, crosslinked with DiSPASO. \nConcentration titrations for ASSB were set from 1  mM to 20 mM, finding an optimal \nperformance plateau at 10 mM with 175 unique residue pairs identified (Figure S3A). Despite \nvarying ASSB concentrations, a consistent loss of crosslinked peptides was observed in the \nenrichment flowthrough, with a maximum loss of 333 links ( Figure S3B). The concentration \nof sodium ascorbate (NaAsc), crucial for efficient click reactions with out compromising the \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n10 \ndisulfide bond in ASSB, was also optimized, reaching a peak efficiency at 20 mM NaAsc with \n242 unique links ( Figure S3C), but with a significant sample loss of 39% after enrichment. \nAdjusting NaAsc to 30  mM aimed to balance click reaction efficiency with minimal side \nreactions. Bead volume optimization for enrichment used ranges from 10 µL to 100 µL of MBS \nbead slurry, with the highest number of detected links (217) at 70 µL (Figure S3E), though not \nentirely preventing sample loss. The sample loss could be reduced by 56% (158 links 20 mM \nASSB vs. 95 links 70 uL bead slurry) but not avoided, even after using 100 uL of bead slurry \n(Figure S3F). \nVarious bead types were tested ( Figure S4A & B ) without markedly reducing sample loss, \nleading to the adoption of a third strategy using disulfide azide agarose beads ( Figure 3C), \nstreamlining the workflow by clicking crosslinked peptides directly to beads.  No additional \npurification steps are needed to avoid unspecific binding by free ASSB molecules and \ntherefore blocking binding sites on the beads. All approaches, despite the methodical \noptimization and strategic shifts, underscored the inherent challenge s in achieving efficient \nand loss-minimized enrichment of crosslinked peptides.  \n \nIn-cell crosslinking using DiSPASO \nThe picolyl azide enrichment strategy was challenged by crosslinking live HeLa cells directly \nin a 6 -well plate using the DiSPASO crosslinker. Unfortunately, this experiment resulted in \nonly 1 crosslink although several attempts were made to push this workflow towards success \n(Figure 6A, grey background).  \n \n \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n11 \nFigure 6: Application of DiSPASO enrichment strategies in increasing sample complexity. A: Spike -in \nexperiment of crosslinked Cas9 in HeLa background. The Cas9 spike-in amount decreased from 0.5 ug \nto 10 ug in a constant background of 100  ug HeLa. A HeLa in -cell crosslinking sample is used as a \n“control” sample. The amount of enriched Cas9 links increases with the amount of Cas9 spike -in. B: \nAnalysis of Monolinks and linear peptides of the Cas9 spike -in experiment. The Monolinks and linear \npeptides of the “control” sample show in general fewer peptides due to the different experimental setup \nof in -cell crosslinking experiments in comparison to a spike -in experiment. The HeLa background \npeptides of the Cas9 spike -in decrease with increasing enrichable Cas9 crosslinks but cannot be \ndepleted completely. C: HEK 293 in -cell crosslinking experiment using DiSPASO for crosslinking and \nDisulfide Azide Agarose beads (DAAB) for click chemistry -based enrichment of crosslinks. The beads \namount is increased from 5 uL beads slurry to 120 uL, the control sample is crosslinked with DiSPASO \nwithout enrichment. With an increasing number of beads, the number of identifiable crosslinks after \nenrichment increases. D: Analysis of Monolinks and linear peptides of the in -cell crosslinking \nexperiment. Linear peptides could not be depleted after crosslink enrichment. \n \nTherefore, the ASSB enrichment workflow was applied to isolated commercial E.coli \nribosomes in a complex HEK 293 cell background to proceed with the crosslink enrichment in \nlive cells (Figure S7A & B). The ASSB enrichment strategy resulted in 14 ribosomal crosslinks \nthat could be enriched from a 1:100 mixture (1  ug crosslinked ribosome spike -in in 100  ug \nHEK 293 cell lysate). This result was not efficient enough for in-cell crosslinking experiments \nand hence, we tried to improve the enrichment procedure by employing Disulfide azide \nagarose beads to directly click the crosslinked peptides to the enrichment handle and the \nbeads in one step. We here titrated the bead amount using crosslinked HEK 293 cells. The \ncells were crosslinked directly in a 10 cm dish using 5 mM DiSPASO resulting in approximately \n9e6 crosslinked cells. The sample was split to each condition equally with a bead amount of \n5-120 uL tested (Figure 6C). 71 unique crosslinks could be detected from the 120  uL bead \nsample, the maximum of crosslink s in this experiment. The linear peptide background could \nnot be reduced across the conditions, possibly due to the unspecific binding of peptides to the \nbeads which is common in bead -based enrichment strategies. The problem of high linear \npeptide background stayed constant across all tested enrichment strategies and experiments, \neven with extensive washing procedures, which pinpoints a systematic problem for this kind \nof enrichment workflow (Figure 6B & D). \nDespite the promising membrane permeability and solubility properties of DiSPASO, mass \nspectrometry experiments yielded low identification rates in in -cell crosslinking studies. To \ninvestigate this discrepancy, we proceeded with confocal microscopy to dire ctly assess the \ncrosslinker's performance in live cells on a visual basis. \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n12 \nMembrane permeability assessment for DiSPASO in HEK cells \n \n \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n13 \nFigure 7: Comparison of DiSPASO and DSBSO uptake during in -cell crosslinking experiments in HEK \n293 cells. A: Confocal microscopy images of DiSPASO during in -cell crosslinking experiments with a \ncrosslink duration of 0 min (Control sample without crosslinking, first left panel), 5min (left second \npanel), 15 min (third left panel), 30min (last panel). The nuclei fluorescenc e signal of DAPI is shown in \nthe upper panel in blue, fluorescence of crosslinked peptides after click reaction to Alexa 488 (green) \nin the middle panel and a merge of both channels on the bottom. The images were taken on an Olympus \nSpinning Disk Confocal microscope (2-024) using a magnification of 40 and a numerical aperture of \n0.75. B: In comparison the DSBSO in-cell experiments were performed in the same way except for the \nfluorophore. DSBSO has an azide as click reaction handle and therefore Alexa 555 ( red) was used to \nvisualise crosslinked peptides. The crosslink duration was set to 0 min (Control sample without \ncrosslinking, first left panel), 5 min (left second panel), 15 min (third left panel), 30 min (last panel). The \nnuclei fluorescence signal of D API is shown in the upper panel in blue, fluorescence of crosslinked \npeptides after click reaction to Alexa 555 (red) in the middle panel and a merge of both channels on the \nbottom. The images were taken on an Olympus Spinning Disk Confocal microscope (2 -024) using a \nmagnification of 40 and a numerical aperture of 0.75. \n \nDiSPASO's performance for in -cell crosslinking was further evaluated through confocal \nmicroscopy, showing rapid uptake by HEK 293 cells, visible in cell nuclei within minutes \n(Figure 7A & Figure S5). The HEK 293 cells, treated with 5mM DiSPASO over 30 minutes, \nrevealed a strong increase in signal within the first 5 minutes, reaching a plateau thereafter \n(Figure 8A). \n \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n14 \n \nFigure 8: Quantitation of crosslink fluorescence signals within nuclei of DiSPASO vs. DSBSO \nmicroscopy images. A: Quantitation of the green Alexa 488 signal of DiSPASO crosslinked peptides. \nThe fluorescence intensity increases fast within the first 5 min after reaching a plateau at 30 minutes. \nEach dot represents a sig nal intensity of crosslinked peptides in a nucleus. B: Nuclei control signal \nintensity of DAPI. The background intensity of DAPI is low in comparison to the intensity of crosslinked \npeptides. C: Quantitation of the red Alexa 555 signal of DSBSO crosslinked peptides. The fluorescence \nintensity increases slowly and reaches a plateau at 30min. D: Nuclei control signal intensity of DAPI. \nThe background intensity of DAPI is also low in comparison to the intensity of crosslinked peptides. \nQuantitation of signal intensities of all images was performed in Fiji ImageJ (version 1.54f).  \n \nThis indicates not only DiSPASO's adeptness at penetrating cellular membranes but also its \nquick reactivity once inside the cell. Such attributes are critical for effective in-cell crosslinking, \nenabling the capture of a broad spectrum of protein-protein interactions in their native cellular \nenvironment. The microscopy images serve as a direct visual affirmation of DiSPASO's \ncapabilities, highlighting the controversy between the visual and the mass spectrometry \nresults. To also evaluate the crosslinker perf ormance compared to other commonly used in -\ncell crosslinking chemistry, we employed Azide -DSBSO19,29 as a benchmark reactant. For a \nfair comparison, HEK 293 cells were treated with 5mM DSBSO in the same manner and \nparallel to the DiSPASO treatment. The difference in the click handle resulted in the use of \ntwo separate fluorescence dyes for each crosslink er. To label DiSPASO, the green dye \nAlexaFluor 488 and for DSBSO the red dye AlexaFluor 555 was used to visualise the \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n15 \nperformance of in-cell crosslinking (Figure 7B). In contrast to DiSPASO, which enters the cell \nvery quickly, DSBSO needs longer incubation times and reaches its maximum performance \nafter 30 min (Figure 8C). The extended reaction time may be attributed to the presence of the \ncharged azide functionality incorporated into the crosslinker that decreases the membrane \npermeability of the crosslinker as shown in Figure 2.  \nOur microscopy data, which validate the predicted chemical properties of DiSPASO, illustrate \nthe crucial balance between hydrophilicity and lipophilicity that governs a compound's \npermeability across biological membranes. While these visual confirmations a lign with \ntheoretical expectations, they also highlight the challenges encountered during sample \npreparation and mass spectrometry analysis, revealing the complexity of translating \ntheoretical advancements into practical applications. \n \nDiscussion \nThe introduction of  DiSPASO in crosslinking mass spectrometry (XL -MS) has opened new \navenues for elucidating protein-protein interactions (PPIs) within cells. DiSPASO has shown \npromising results via confocal microscopy, particularly with its successful cellular uptake and \nquick reactivity once inside the cell, highlighting its potential for in -depth cellular studies as \nwell as revealing hurdles during mass spectrometry analysis.  \nIn general, cleavable crosslinkers have labile bonds that break at lower energies than the \npeptide backbone, producing a distinct fragmentation pattern useful for analysing complex \nmixtures. However, an excess of cleavage sites can reduce the identification rate, as observed \nwith DiSPASO in our study. Consequently, one of the two signal doublets resulting from \ncrosslinker cleavage may be absent, possibly due to further fragmentation or decreased signal \nintensity caused by an excess of fragment species in th e spectra 10,35,36. In contrast to the \nanticipated cleavage pattern, our findings reveal that DiSPASO exhibits unexpected additional \ncleavage sites beyond those expected for DSBSO, resulting in the formation of extra \nfragments. While it has been documented that carbon−sulfur bonds in benzyl mercaptans can \nrapidly dissociate under specific conditions, such as UVPD irradiation at 213 nm and 266 \nnm37,38, this phenomenon was previously limited to certain wavelengths. Interestingly, recent \nstudies have shown that C−S bond -selective photodissociation with 213 nm is augmented \nwhen sulfur is absent from an aromatic system by one  methylene group (sp3 carbon) but \nhampered when sulfur is directly attached to a sp2 carbon. Surprisingly, our experiments \nindicate that this cleavage mechanism occurs even during standard HCD fragmentation. \nDespite the challenges of complex fragmentation patterns and the need for refining data \nanalysis, these findings offer valuable insights that drive further advancements in the field. \nThe development of DiSPASO marks a significant step forward in enhancing cellular PPI \nmapping with high specificity and efficiency. However, the challenges encountered in \ntranslating its theoretical advantages into practical utility reveal a critical discrepancy that \nunderscores the need for ongoing refinement in crosslinker te chnology. DiSPASO highlights \nthe intricate balance between chemical innovation and biological functionality, emphasizing \nthat advancements in crosslinker design must address current limitations to fully realize the \npotential of XL-MS in studying cellular mechanisms. While DiSPASO represents progress in \nPPI analysis, its limitations necessitate a cautious approach and highlight the complexity of \ndeveloping effective crosslinking tools. Continuous innovation in structural design, particularly \nin simplifying c rosslinkers and reducing potential cleavage sites, is essential. Additionally, \nadvancements in computational tools and crosslinking search algorithms are crucial to \novercoming challenges related to the search space in crosslinking data, enhancing the \nperformance of non -cleavable crosslinkers for in -cell studies. These improvements will \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n16 \nsimplify data analysis and pave the way for more effective, streamlined XL -MS applications, \nultimately bringing us closer to accurate and efficient mapping of PPI networks. Further studies \ninvolving different enrichment handles and reactive sites are ongoi ng to expand the \ncapabilities of DiSPASO and similar crosslinkers. \nData availability \nThe mass spectrometry proteomics data have been deposited to the ProteomeXchange \nConsortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository39 \nwith the dataset identifier PXD056091. \n \nReferences \n1. Matzinger, M. & Mechtler, K. 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Nucleic Acids Res. 50, D543–D552 (2022). \nAcknowledgements \nThis work was supported by the infrastructure funding 4 th call 2022/01 (AT -SCP) of the \nAustrian Research Promotion Agency (FFG) and the project LS20-079 of the Vienna Science \nand Technology Fund (WWTF). This work was further funded by the ESPRIT program project \nnumber ESP 566 (Grant -DOI 10.55776/ESP566), P35045 -B project (Grant -DOI \n10.55776/P35045) and the F 8801 -B Meiosis project (Grant -DOI 10.55776/F88) of the \nAustrian Science Fund (FWF). All LC -MS/MS analyses in Vienna were performed on the \nVienna BioCenter Core Facilities instrument pool.  We thank the V ienna Biocentre BioOptics \nfacility for help and advice with microscopy imaging. Synthesis was performed at the Institute \nof Organic Chemistry of the University of Vienna. Funding from the Austrian Academy of \nSciences (DOC Fellowship to B.R.B.) is acknowledged. We thank the University of Vienna for \nits generous support. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n20 \nThis research was funded in whole, or in part, by the Austrian Science Fund (FWF). For open \naccess, the author has applied a CC BY public copyright license to any Author Accepted \nManuscript version arising from this submission. \n \nCompeting interest statement \nThe authors declare no competing interest.  \n \nEthics approval and consent to participate \nNot applicable. \n \nConsent for publication \nNot applicable. \n \nSupplementary information \nThe online version contains supplementary material as listed below. \n \nSupplement \n \nOrganic Synthesis ................................ ................................ ................................ .............. 3 \nGeneral Information ................................ ................................ ................................ ........... 3 \nSynthesis of DiSPASO ................................ ................................ ................................ ...... 4 \nGeneral Procedure 1: Synthesis of Aryl Bromide (GP-1) ................................ ................ 5 \nGeneral Procedure 2: Reduction of Esters (GP-2) ................................ ......................... 6 \nGeneral Procedure 3: Halogenation of Alcohols (GP-3) ................................ ................. 6 \nGeneral Procedure 4: Synthesis of Key Intermediate S5 (GP-4) ................................ .... 7 \nGeneral Procedure 5: Sonogashira Cross-Coupling (GP-5) ................................ ........... 8 \nGeneral Procedure 6: Global Deprotection (GP-6) ................................ ......................... 9 \nGeneral Procedure 7: Synthesis of NHS Protected Precursor S8 (GP-7) ..................... 10 \nGeneral Procedure 8: Synthesis of NHP Protected Precursor (GP-8) .......................... 11 \nGeneral Procedure 9: Synthesis of DiSPASO and DiPPASO (GP-9) ........................... 13 \nNMR Spectra ................................ ................................ ................................ ................... 15 \nMethods ................................ ................................ ................................ ............................. 25 \nReagents ................................ ................................ ................................ ......................... 25 \nPeptide Synthesis ................................ ................................ ................................ ............ 25 \nCrosslinking reaction for Cas9 ................................ ................................ ......................... 26 \nSingle peptide crosslinking ................................ ................................ ..............................  26 \nIn-Solution Digest ................................ ................................ ................................ ............ 26 \nClick reaction using Azide-S-S-biotin ................................ ................................ ............... 26 \nCrosslinked peptide enrichment................................ ................................ ....................... 27 \nRibosome crosslinked with DiSPASO ................................ ................................ .............. 27 \nSensitivity experiment using picolyl azide as click reagent................................ ............... 28 \nIn-cell crosslinking of HEK and HeLa cells using DiSPASO ................................ ............. 29 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint \n\n21 \nCrosslink enrichment using Disulfide Azide Agarose beads (DAAB) ................................  29 \nSample preparation for confocal microscopy of crosslinked HEK cells ............................ 30 \nConfocal microscopy procedure ................................ ................................ ...................... 30 \nRelative quantitation of fluorescence signals ................................ ................................ ... 31 \nMass spectrometry ................................ ................................ ................................ .......... 31 \nData analysis ................................ ................................ ................................ ................... 31 \nSoftware adjustments ................................ ................................ ................................ ...... 32 \nSurface area plot creation................................ ................................ ................................  33 \nSupplemental figures ................................ ................................ ................................ ........ 33 \nReferences ................................ ................................ ................................ ........................ 41 \n \n \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2024. ; https://doi.org/10.1101/2024.11.05.621843doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}