{"paper_id":"0c2ee7f7-74da-4110-ad7b-ce77b758a51c","body_text":"1 \nAffinity-Based Interactome Mapping of Inositol \nPyrophosphates Reveals 4/6 -PP-InsP5–Binding Proteins \nin Plants \nKevin Ritter1, Verena Gaugler2, Sara Christina Stolze 3, Riya Ghosh4, Akhila Jayamon4, Felix \nWollensack1, Debabrata Laha4, Hirofumi Nakagami3, Gabriel Schaaf2, Henning Jacob Jessen1* \n1 Faculty of Chemistry and Pharmacy, Institute of Organic Chemistry, and CIBSS-Centre for \nIntegrative Biological Signaling Studies, Albert-Ludwigs University Freiburg, 79104 Freiburg, \nGermany \n2 Department of Plant Nutrition, Institute of Crop Science and Resource Conservation, \nRheinische Friedrich-Wilhelms-Universität Bonn, 53115 Bonn, Germany \n3 Protein Mass Spectro metry, Max Planck Institute for Plant Breeding Research, 50829 \nCologne, Germany \n4 Department of Biochemistry, Division of Biological Sciences, Indian Institute of Science \n(IISc), Bengaluru 560012, India \n*Email: henning.jessen@oc.uni-freiburg.de \nAbstract \nInositol pyrophosphates (PP-InsPs) are central regulators of eukaryotic signaling events. While \ncertain PP-InsP isomers have been conclusively linked to the regulation of phosphate \nhomeostasis through interaction with SPX domain containing proteins in plants, the functions \nof the recently discovered  isomer 4/6-PP-InsP5 remain largely unknown. Here, we employed \ntwo complementary affinity-based strategies – a matrix approach and a photoaffinity probe – to \nsystematically identify 4/6 -PP-InsP5-binding proteins in Arabidopsis thaliana . The two \nmethods yielded partially overlapping protein sets, with photoaffinity enrichment likely \ncapturing additional transient and/or weak interactions . Moreover, competition experiments \nwith different isomers were applied to obtain information about potential isomer -specific \ninteractions. As a proof-of-concept, one candidate interactor ( FHA domain-containing protein \nAtFHA2) was shown to bind 4 -PP-InsP5 in vitro with substantially higher affinity than InsP6. \nThus, besides the SPX domain, FHA domain containing proteins, of which 18 exist in \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 It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 2 \nArabidopsis, are potentially regulated by inositol pyrophosphates. More generally, our findings \nreveal a diverse protein network associated with 4/6-PP-InsP5 and establish a versatile platform \nfor dissecting its biological roles in plants and other organisms. \n1 Introduction \nInositol phosphates (InsPs) are a diverse class of highly charged intracellular signaling \nmolecules derived from myo-inositol (1), a cyclohexane hexol with a distinct stereochemistry \nincluding an internal mirror plane, which classifies it as a meso-compound.[1–3] The addition of \nphosphate and diphosphate groups at different hydroxyl positions generates a vast array of \nregioisomers and enantiomers  through desymmetri zation.[4,5] Among those, inositol \npyrophosphates (PP-InsPs) represent a densely phosphorylated subset that plays a critical role \nin cellular regulation, influencing processes such as phosphate homeostasis, energy \nmetabolism, and stress responses across organisms.[2,3,6,7] \n \nFigure 1: Chemical structures of selected inositol pyrophosphates. a) 5-PP-InsP5 (2) and 1,5-(PP)2-InsP4 (3), the two most \nextensively studied PP-InsPs in mammalian and plant systems. b) 4-PP-InsP5 (4) and 6-PP-InsP5 (5) are enantiomers and were \nrecently identified in plants, mammalian cells, and other eukaryotic species, while the exact configuration remains unknown.  \nResearch has primarily focused on 5 -PP-InsP5 (2) and 1,5 -(PP)2-InsP4 (3) in mammals  and \nplants, but recent studies demonstrated that other PP-InsP isomers (see Figure 1) are widespread \nand more abundant than previously thought. 6-PP-InsP5 (5) was initially believed to be unique \nto Dictyostelium discoideum, where it is the predominant PP-InsP5 isomer.[8,9] However, recent \nstudies have identified 4/6 -PP-InsP5 (4/5) in various eukaryotic systems, including plants, \npatient-derived peripheral blood mononuclear cells (PBMCs), and mouse colon and heart \ntissues[9–12]. These findings were obtained using capillary electrophoresis –mass spectrometry \n(CE-MS) with heavy isotope labeled internal references, confirming its occurrence across \ndiverse biological systems [10,11]. Notably, in all studied land plants and PBMCs, 4/6-PP-InsP5 \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 3 \n(4/5) was detected at levels comparable to or exceeding those of 5 -PP-InsP5 (2), suggesting a \nmore prominent role than previously assumed. [9,11,12] Since CE -MS does not discriminate \nbetween enantiomers, the signal could arise from 4-PP-InsP5, 6-PP-InsP5 (4 or 5), or both. For \nclarity, we refer to them collectively as 4/6-PP-InsP5 (4/5). The detection of this isomer beyond \nD. discoideum challenges long-standing assumptions about PP-InsP metabolism and highlights \nthe need to reassess the functional significance of 4/6 -PP-InsP5 (4/5) in eukaryotic signaling \npathways. \nIn Arabidopsis thaliana , the inositol polyphosphate multikinase AtIPK2 α  and AtIPK2β \nphosphorylate InsP6 (6) to generate 4/6 -PP-InsP5 (4/5) in vitro.[12] Together they regulate the \ncellular levels of 4/6-PP-InsP5 (4/5) in planta. Notably, these kinases play a critical role in heat \nstress acclimation, as their disruption leads to impaired expression of heat shock proteins and \nreduced thermo-tolerance.[12] The evolutionary conservation of this function is supported by \nfindings in Marchantia polymorpha , where an IPMK homolog contributes to heat stress \nresponses, suggesting an ancient role of 4/6 -PP-InsP5 (4/5) in environmental adaptation. [12] \nBeyond heat stress, PP -InsPs regulate phosphate homeostasis via SPX -domain-containing \nproteins, which mediate phosphate starvation responses. [13] Additionally, certain NUDIX \nhydrolases selectively degrade 4 -PP-InsP5 (4), suggesting an isomer -specific regulatory \nmechanism.[14,15] Taken together, these insights highlight the need to further investigate the \nspecific roles of 4/6-PP-InsP5 (4/5) in plant signaling networks, particularly its potential impact \non stress adaptation. \nDespite the growing recognition of 4/6 -PP-InsP5 (4/5) as a functional signaling molecule, its \nprotein interactome remains largely unexplored. Previous affinity enrichment studies have been \nlimited to 5 -PP-InsP5 (2) and 1,5 -(PP)2-InsP4 (3) in non -plant systems, expanding our \nunderstanding of PP-InsP interactomes and providing new candidates for functional studies in \nyeast and mammalian cells. [16–18] In Arabidopsis, affinity enrichment experiments have so far \nbeen conducted exclusively for 5 -PP-InsP5 (2), using an Affi-Gel method, in which a  \nnonhydrolyzable 5-PCP-InsP5 (7) analog is immobilized on a resin matrix to enable selective \nprotein binding. [14,15] Given the structural differences between PP -InsP isomers and their \npotential for distinct protein interactions,[6,19] a targeted approach to characterize the interactors \nof 4/6-PP-InsP5 (4/5) is necessary. \nTo address this gap, we applied complementary affinity-based enrichment strategies to identify \n4/6‑PP‑InsP5-binding proteins in the flowering plant Arabidopsis thaliana. By combining a \nmatrix-based approach with a photoaffinity labeling method, we systematically mapped 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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 4 \nligand’s interactome. Our study provides a robust methodological framework for investigating \n4/6‑PP‑InsP5 signaling in plants and beyond. \n2 Results & Discussion \nSynthesis of Amino-PEG-4/6-PCP-InsP5 \nTo enable the selective enrichment of 4/6 -InsP5-binding proteins, we developed a modular \ninositol pyrophosphate analog bearing a terminal amine suitable for covalent modification or \nresin attachment. The resulting compound, Amino -PEG-4/6-PCP-InsP5 (14), consists of a \nmethylene bisphosphonate (PCP) at either the 4 - or 6 -position of myo-inositol (1), and a \npolyethylene glycol (PEG) linker with a terminal amine installed on the opposite phosphate. \nThat is, when the PCP group is located at position 4, the PEG linker is attached at position 6, \nand vice versa. As the 4- and 6-positions are enantiotopic, and it is not clear whether 4 - or 6-\nPP-InsP5 (4 or 5) is the biologically relevant isomer, the compound was synthesized and used \nas a racemic mixture of 4- and 6-PCP-isomers. \nOur compound design builds on an affinity enrichment strategy established by Wu et al., who \nimmobilized a nonhydrolyzable methylene bisphosphonate (PCP) analog of 5‑PP‑InsP5 (2) on \nAffi‑Gel resin for pull -down experiments .[16] PCP analogs are chemically stabilized \ndiphosphate mimics that preserve the geometry and charge of native PP‑groups while resisting \nhydrolysis and eliminating phosphoryl transfer, making them powerful tools for probing \nPP‑InsP signaling.[20,21] Other stabilized diphosphate mimics, including α‑phosphonoacetic acid \n(PA) esters and difluoro‑substituted analogues, have also been developed to retain non‑covalent \nrecognition while blocking phosphoryl transfer, but they have not yet been used in enrichment \nworkflows.[22,23] To address this gap, we synthesized a PCP-containing analog of 4/6‑PP‑InsP5 \n(4/5) designed for both resin coupling and photoaffinity labeling.   \nThe starting point for this synthesis was a previously established strategy for the regioselective \nfunctionalization of the 4 - and 6-positions of myo-inositol (1) (see Scheme 1 ).[24] In the first \nstep, myo-inositol (1) was protected as its orthoformate using triethyl orthoformate under acidic \nconditions. Selective silylation at position 2 with TBSCl and a sterically hindered base (2,6 -\nlutidine) enabled differentiation between axial and equatorial hydroxyl groups.[24] Allyl groups \nwere then introduced at positions 4 and 6 to allow for orthogonal deprotection in later steps, \naffording the bis-allylated inositol derivative 8. \nAcid treatment removed both the orthoformate and the TBS group, releasing hydroxyl groups \nat positions 1, 2, 3, and 5. These were phosphorylated using a standard phosphoramidite \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 5 \nprotocol with dibenzyl phosphoramidite and 4,5-dicyanoimidazole (DCI) as activator, followed \nby oxidation with meta-chloroperbenzoic acid ( mCPBA).[4,25,26] The resulting tetraphosphate \nintermediate 10 was then subjected to PdCl2-mediated deallylation, releasing hydroxyl groups \nat positions 4 and 6. [26] As prolonged reaction times led to phosphate migration, the reaction \nwas carefully timed and monitored via 31P-NMR. Following phosphate migration, the resulting \nregioisomeric mixtures cannot be resolved, making it essential to prevent their formation. \nThe PCP group was introduced using PCP -phosphoramidite ( S2) synthesized according to \nHostachy et al.[27] As the substitution occurs at either the 4 - or 6-position of the inositol, and \neach phosphorylation step creates a stereogenic center at phosphorus upon oxidation, a total of \nfour stereoisomers are formed – two diastereomers, each present as a pair of enantiomers. Two \ndistinct species were observed both by 31P-NMR spectroscopy, with signals in a ratio of \napproximately 2:3, and by LC -MS, which showed closely eluting peaks of identical mass. \nSeparation of the diastereomers by chromatography was not attempted,  as the stereogenic \ncenters at phosphorus collapse upon global deprotection. Their transient formation nevertheless \nconfirmed the successful and selective incorporation of the PCP group. \nThe final phosphate diester, bearing a PEG linker with a terminal primary amine, was installed \nat the remaining free hydroxyl group using the phosphoramidite approach. The PEG -\nphosphoramidite was synthesized following the procedure reported by Wu et al. [16] Upon \noxidation, the phosphorus atom of the newly introduced group becomes stereogenic, adding an \nadditional layer of stereochemical complexity to the molecule. Consequently, prior to global \ndeprotection, up to eight stereoisomers – corresponding to four pairs of enantiomers – are \ntheoretically possible. However, because of signal overlap and the unequal formation of \nindividual species, the resulting diastereomers could not be fully resolved by NMR or LC-MS. \nSubsequent catalytic hydrogenation over Pd/C effe cted global debenzylation in a single step, \neliminating the complex stereochemical mixture generated earlier . The target compound, \nAmino-PEG-4/6-PCP-InsP5 (14), was obtained as a mixture of the two enantiomers with \nsubstitution at either the 4 - or 6-position of myo-inositol. This molecule served as a precursor \nfor both affinity enrichment strategies described in this study. The free amine allowed direct \ncoupling to NHS -activated Affi-Gel resin, as previously shown for  the PCP analog of  5-PP-\nInsP5 (2),[16] and was also compatible with trifunctional photoaffinity linkers . Applying this \nstrategy to 4/6-PP-InsP5 (4/5) provided the basis for identifying stereoisomer -specific protein \ninteractors in plants. \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 6 \n \nScheme 1: Synthesis of Amino-PEG-4/6-PCP-InsP5 (14). Reagents and conditions: (a) NaH (2.6 eq), allyl bromide (2.6 eq), \nNaI (cat.), DMF, 0°C to rt, overnight. (b) Bn-PA (7.0 eq), DCI (7.0 eq), DMF, rt, 2h; then mCPBA (7.0 eq), 0°C to rt, 10 min. \n(c) PdCl2 (2.0 eq), MeOH, rt, 2h. (d) PCP-PA (1.2 eq), DCI (2.0 eq), CH2Cl2 rt, 1.5h; then mCPBA (2.0 eq), 0°C to rt, 10 min. \n(e) Fm-DiPA (6.2 eq), PEG-linker-alcohol (6.2 eq), ETT (6.2 eq); then 12 (1.0 eq), ETT (3.0 eq), CH 2Cl2 rt; then mCPBA \n(3.0 eq), 0°C to rt, 10 min. (f) H2 (30 bar), Pd/C (3.0 eq), NaHCO3 (13.0 eq), tBuOH/H2O (40:7), rt, 21 h. Abbreviations: Bn-\nPA = Bis-benzyl-N,N-diisopropylamino phosphoramidite; DCI = 4,5-Dicyanoimidazol; ETT = 5-Ethylthio-1H-tetrazole; Fm-\nDiPA = 9H-fluoren-9-ylmethyl-bis(N,N-diisopropylamino) phosphordiamidite. mCPBA = meta-chloroperoxybenzoic acid; PA \n= phosphoramidite; PCP = methylenebisphosphonate. \nSynthesis of a Trifunctional-Photoaffinity Compound \nPhotoaffinity capture enables covalent crosslinking of ligand -binding proteins upon UV \nactivation, allowing detection of transient or low -affinity interactions that are not readily \ncovered by conventional pull -down approaches. [28,29] To apply this strategy to 4/6 -PP-InsP5 \n(4/5), a suitable linker must combine three essential features: a photoreactive group for UV -\ninduced crosslinking, a biotin-based tag for streptavidin-mediated enrichment, and an activated \nester for coupling to the amino-functionalized probe. \nBiotin and desthiobiotin both form strong non-covalent interactions with streptavidin, enabling \nefficient recovery of labeled protein complexes via immobilized streptavidin matrices, \nincluding agarose or magnetic beads. [28,30] A commonly used linker that fulfills these \nrequirements is Sulfo-SBED (Thermo Fisher Scientific), which integrates an aryl azide, biotin, \nand a cleavable disulfide bridge. However, this reagent is costly and incompatible with reducing \nagents such as DTT, which are often used in lysate preparations. \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 7 \nTo overcome these limitations, we designed a custom photoaffinity linker that retained the \nessential functional elements of Sulfo -SBED but replaced the disulfide bridge with a stable \nbackbone and featured desthiobiotin instead of biotin. The custom linker (19, see Scheme 2A) \nwas synthesized in seven steps, starting from a commercially available Boc -protected lysine \nmethyl ester ( 15). In the first step, the photoreactive aryl azide was introduced via coupling \nwith 4 -azidobenzoic acid under standard peptide c oupling conditions (HOBt, EDC I, NEt₃), \naffording intermediate 16 in 94% yield. After basic hydrolysis of the methyl ester, the resulting \ncarboxylic acid was coupled with aminohexanoic methyl ester ( S8) to give compound 17 in \n70% yield. Aminohexanoic methyl ester (S8) was synthesized separately following a reported \nprocedure.[31] Subsequent Boc deprotection using trifluoroacetic acid (TFA) afforded the free \namine, which was immediately subjected to a third peptide coupling with desthiobiotin, yielding \nintermediate 18 (68% yield). Final hydrolysis of the methyl ester and in situ activation with \nDCC in DMF furnished the sulfonated NHS ester 19. The sulfonated NHS ester  ensured \naqueous solubility, as the final coupling to the Amino-PEG-4/6-PCP-InsP5 probe (14) had to be \ncarried out in water. \nConjugation of the photoaffinity linkers to the Amino -PEG-4/6-PCP-InsP5 probe ( 14) was \nperformed in aqueous sodium bicarbonate buffer under mild conditions, yielding the two fully \nfunctionalized capture reagents depicted in  Scheme 2B. Conjugation with the custom linker \nafforded compound 20, while coupling with the commercially available Sulfo -SBED linker \nprovided compound 21, both  isolated in different protonation degrees with TEAA as \ncounterions after purification . Both reagents were obtained as racemates and were applied in \nphotoaffinity pulldown experiments to identify 4/6-PP-InsP5-binding proteins. \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 8 \n \nScheme 2: (A) Synthesis of custom-photoaffinity linker (17). (B) Structures of the racemic photoaffinity reagents with custom \n(20) and commercial linker (21). Reagents and conditions: (a) 4-azodibenzoic acid (1.1 eq), HOBt (1.2 eq), EDCI (1.2), NEt3 \n(3.5 eq), CH2Cl2, rt, overnight. (b) aq. NaOH (2.0 eq), MeOH, 0°C, 30 min; then S8 (1.1 eq), HOBt (1.2 eq), EDCI (1.2 eq), \nNEt3 (3.5 eq), rt, overnight. (c) TFA (33 % v/v), CH2Cl2, rt, 90 min; then desthiobiotin (1.1 eq), HOBt (1.5 eq), EDCI (1.5 eq), \nNEt3 (3.0 eq), DMF, rt, overnight. (d) aq. NaOH (1M), MeOH, 0°C, 90 min; then sulfo-NHS (1.0 eq), DCC (3.0 eq), rt, 48h. \nAbbreviations: HOBt = N-Hydroxybenzotriazole; EDCI = 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide; DCC = N,N′-\nDicyclohexylcarbodiimid. \nAffinity Enrichment and Proteomic Analysis \nRoot and shoot  material of Arabidopsis was prepared as previously described.[14] Tissue was \nground in liquid nitrogen and extracted with magnesium -containing lysis buffer. DTT was \nincluded for Affi-Gel and custom linker ( 20) experiments but omitted for commercial linker \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 9 \n(21) to prevent disulfide reduction. Lysates were clarified by centrifugation and directly used \nfor enrichment. \nTo probe non-covalent interactions, amino -functionalized PCP analogs of 4/6 -PP-InsP5 (4/5) \nwere immobilized on NHS -activated agarose beads, according to Furkert et al. [32] Negative \ncontrol matrices contained immobilized inorganic phosphate coupled to the same linker. Beads \nwere incubated with clarified lysates to allow equilibrium binding, then extensively washed. \nBound proteins were eluted with 20 mM InsP6 (6) (elution fraction), while remaining proteins \nwere subjected to on -bead trypsin digestion (on -bead fraction). Both fractions were analyzed \nby LC -MS/MS. The workflow was adapted from Wu et al. and Schneider et al. [14,16] \nComparative enrichment versus control matrices revealed candidate interactors. \nIn parallel, a photoaffinity-based approach was used to covalently capture protein interactions \n(see Figure 2), adapted from Haas et al.[33] Two biotinylated capture compounds were applied, \nboth comprising a non-hydrolyzable analog of 4/6‑PP‑InsP5 (4/5), a photoreactive group, and a \nsorting handle. The design and synthesis of both linker systems are described in detail above \n(see Scheme 2). \nPrior to probe addition, lysates were either left untreated or pre -incubated with a 300 -fold \nexcess of soluble competitors – enantiopure 4-PP-InsP5 (4) or 6-PP-InsP5 (5) – to assess binding \nspecificity and potential enantioselectivity in binding. Capture compounds were added and \nincubated at 4 °C, followed by UV irradiation (365 nm, 30 min) to induce covalent crosslinking. \nResulting complexes were isolated using streptavidin magnetic beads. DTT was included with \nthe custom linker to stabilize interactions, but omitted with the commercial linker to preserve \nthe disulfide bridge. After washing, beads were stored at −80 °C. Candidate interactors were \ndefined as proteins enriched in non-competed samples compared to competitor-treated controls, \nindicating specific or stereoselective binding. \nProteins from both enrichment approaches were processed using a unified proteomics \nworkflow. Trypsin digestion was performed either on-bead (photoaffinity and Affi-Gel beads) \nor in-solution (Affi-Gel eluates), followed by desalting and LC -MS/MS analysis on  Orbitrap \nmass spectrometers. Protein identification and label -free quantification were performed with \nMaxQuant, and statistical analysis of protein enrichment was performed in Perseus using a \nconsistent threshold (S 0 = 1, FDR = 0.05). [34,35] Proteins with a log 2 fold change > 2 were \nconsidered candidate interactors. Quantitative filtering was set to a minimum of three valid \nvalues per condition for Affi-Gel datasets and two for photoaffinity experiments. \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 10 \n \nFigure 2: Schematic overview of the photoaffinity-based enrichment workflow used to identify 4/6-PP-InsP5-binding proteins \nin Arabidopsis thaliana. Top left: Sample preparation from root and shoot tissue. Top center: Structure of the photoaffinity \nreagent featuring a photoreactive group, a sorting function, and a selection function. Center panels: Parallel workflows with \n(red) and without (blue) competition control, including incubation with the photoaffinity reagent, UV crosslinking, and \nenrichment with streptavidin magnetic beads. Right: Analysis and evaluation steps involving on-bead digestion, LC-MS/MS, \nand data interpretation using volcano plots. Figure was created with Biorender.com. \nGene Ontology (GO) enrichment analysis was performed using g:Profiler (default parameters), \nfocusing on categories related to phosphatidylinositol metabolism and inositol phosphate \nsignaling. Shown terms represent Driver Terms as defined by g:Profiler.[36]  A condition-specific \nmatrix listing all proteins with log₂ enrichment > 2 in at least one condition, including gene IDs, \nannotations, and fold changes, is provided in the Supporting Information. Data processing and \nvisualization were supported by custom R  scripts (partly generated via ChatGPT -4o) used to \nstructure the matrix, apply filtering, and generate volcano plots, UpSet diagrams, and GO-term \ncharts. \nAffinity Enrichment: Comparative Results and Key Insights \nComparative analysis of the enrichment datasets provides an integrated view of the 4/6 -PP-\nInsP5 interactome in Arabidopsis, revealing how tissue type, enrichment strategy, and \ncompetition isomer influence the captured protein subsets. \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 11 \nAffinity-based enrichment using Affi-Gel matrices yielded a moderate but specific set of 4/6 -\nPP-InsP5-binding proteins, with about 40 –100 proteins enriched per condition ( see Figure 3). \nIn addition to direct on-bead digestion, an elution fraction was generated by releasing proteins \nwith InsP6 (6). The enrichment profiles showed a combination of proteins consistently detected \nacross all conditions and others that appeared only in specific tissues or fractions. Eight proteins \nwere reproducibly enriched in eve ry condition  – AT1G07310.1 (CaLB -domain protein), \nAT1G10900.1 (phosphatidylinositol -5-kinase), AT1G12380.1 (uncharacterized protein), \nAT1G31440.1 (SH3-domain protein), AT1G47550.1 (SEC3A), AT3G03790.1 (ankyrin/RCC1 \nrepeat protein), AT3G22170.2 (FHY3), and AT4G25550.1 (cl eavage and polyadenylation \nfactor) – suggesting that they represent stable, high-affinity interactors. \nOverlap analysis showed that on -bead fractions contained a broader range of interactors than \neluates, with 17 proteins shared between root and shoot on -bead samples but only two in \neluates. This indicates that on -bead fractions mainly enrich stronger or mo re stable binders, \nwhereas eluates capture weaker or more transient associations. Several proteins were unique to \nindividual conditions, with the shoot on -bead fraction containing the largest number of \nexclusive hits. GO -term analysis revealed significant enrichment of categories related to \nphosphatidylinositol metabolism and inositol phosphate signaling, consistent with the expected \nbiological roles of 4/6-PP-InsP5 (4/5). Collectively, these results demonstrate that the Affi-Gel \napproach captures a selective set of high-affinity interactors with low background, providing a \nreliable platform for validation and future mechanistic studies. \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 12 \n \nFigure 3:  Analysis of Affi-Gel datasets from Arabidopsis thaliana samples. a,b) V olcano plots showing significantly enriched \nproteins (FDR < 0.05, S 0 = 1) from Affi -Gel on-bead fractions of root (panel a) and shoot (panel b) samples. Highlighted \nproteins (blue) were selected based on high enrichment scores and/or their potential relevance to inositol phosphate signaling \nand phosphoinositide metabolism, including: a) AT5G07370.4 (IPK2α; inositol polyphosphate kinase 2 alpha), AT1G13960.2 \n(WRKY4; WRKY DNA -binding protein 4), AT1G22620.1 (ATSAC1; phosphoinositide phosphatase family prot ein), \nAT1G43670.1 (inositol monophosphatase family protein), and AT3G22170.2 (FHY3; far -red elongated hypocotyls 3).  b) \nAT3G22170.2 (FHY3; far-red elongated hypocotyls 3), AT1G47550.2 (SEC3A; exocyst complex component), AT2G01670.1 \n(NUDT17; nudix hydrolase homolog 17), AT4G30935.1 (WRKY32; WRKY DNA -binding protein 32), and AT1G31440.1 \n(SH3 domain-containing protein). c) UpSet plot illustrating the overlap of enriched proteins across root and shoot samples. d) \nGene Ontology (GO) analysis of enriched proteins from root and shoot samples, showing Driver Terms identified by g:Profiler. \nFor this analysis, protein lists from on -bead and elution fractions were combined. Categories are grouped into molecular \nfunction (blue) and biological process (red). \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 13 \nPhotoaffinity-based enrichment revealed a broader and more variable interactome than the Affi-\nGel approach, reflecting the ability of covalent crosslinking to stabilize transient associations \nand retain weak binders that would otherwise be lost during washing. Photoaffinity probes may \nalso label protein complexes associated with direct interactors, further expanding the apparent \ninteractome. In roots, 180–380 proteins were enriched per condition (see Figure 4) and in shoots \n90–490 ( see Figure 5). Across all  experiments, the custom -designed linker (compound 20) \nconsistently yielded more enriched proteins than the commercial Sulfo -SBED reagent \n(compound 21). This difference likely reflects the inclusion of DTT in the custom linker \nexperiments, which helped preserve protein integrity, whereas DTT was omitted with the \ncommercial linker to avoid cleavage of its disulfide bridge. Additionally, linker design features \nsuch as crosslinking efficiency, spatial arrangement of reactive groups, and target accessibility \nmay also have contributed to the broader interactome coverage observed with the custom linker. \nThese probe -specific properties should be considered when interpreting the data and when \nplanning future validation experiments. \nCompetition experiments consistently showed that free 6 -PP-InsP5 (5) displaced a broader set \nof proteins than 4 -PP-InsP5 (4), suggesting a more diverse interactome. In root samples, the \ncustom linker yielded 376 proteins displaced by 6-PP-InsP5 (5) compared to 285 by 4-PP-InsP5 \n(4), and in shoots the difference was even greater (493 vs. 198). With the commercial linker, \nthe same trend was seen in shoots (206 vs. 90), whereas in roots 4 -PP-InsP5 (3) displaced \nslightly more proteins than 6 -PP-InsP5 (4) (177 v s. 164). These results support the often -\ncontested view that PP -InsP5 isomers display distinct binding profiles, consistent with their \nhaving potentially different biological roles. Despite substantial overlap, each isomer also \nrecruited unique subsets of interactors, indicating that 4-PP-InsP5 (4) and 6-PP-InsP5 (5) engage \nboth with shared and specific binding partners under the tested conditions. \nNotably, both root and shoot samples showed substantial but incomplete overlap of proteins \nacross enrichment conditions, indicating that linker design, competition strategy, and tissue \ncontext shape the captured interactome. Unique subsets were detected in individual competition \nsetups, with the largest proportion in custom linker 6-PP-InsP5 (5) experiments for both tissues. \nThese findings underline that candidate interactors must be interpreted with caution and require \nthorough follow -up analysis.  Importantly, proteins identified by affinity -based enrichment \nshould not be assumed to always reflect physiologically relevant interactions. The loss of \ncellular compartmentalization during extraction may give rise to artefactual associations, and \nproteins that are part of multi-protein assemblies may co-purify without directly binding to the \nligand. \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 14 \n \nFigure 4: Analysis of photoaffinity -enriched datasets from Arabidopsis thaliana  root samples. a,b) Vo l c a n o  p l o t s  o f  \nsignificantly enriched proteins (FDR < 0.05, S0 = 1) from competition experiments with free 4-PP-InsP5 (panel A) or free 6-\nPP-InsP5 (panel B) as competitors.  Highlighted proteins (blue) were selected based on high enrichment scores and/or their \npotential relevance to inositol phosphate signaling and phosphoinositide metabolism, including: a) AT4G00670.1 (Remorin \nfamily protein), AT1G48920.1 (NUC -L1; nucleolin like 1), AT3G12360.1 (ITN1; ankyrin repeat family protein), \nAT1G65280.1 (DNAJ heat shock N-terminal domain-containing protein), and AT3G20650.1 (mRNA capping enzyme family \nprotein). b) AT5G51280.1 (DEAD -box protein, putative), AT3G20650.1 (mRNA capping enzyme family protein), \nAT1G65280.1 (DNAJ heat shock N -terminal domain -containing protein), AT1G20920.1 (P -loop containing nucleoside \ntriphosphate hydrolases superfamily protein), and AT3 G18610.1 (NUC-L2; nucleolin like 2).  c) UpSet plot illustrating the \noverlap of enriched proteins across all root samples from photoaffinity enrichment experiments, including datasets from both \ncustom and commercial linker experiments with 4-PP-InsP5 and 6-PP-InsP5 as competitors. d) Gene Ontology (GO) analysis \nof enriched proteins from root samples, showing Driver Terms identified by g:Profiler. For this analysis, all root sample datasets \nwere combined. Categories are grouped into molecular function (blue) and biological process (red). \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 15 \n \nFigure 5: Analysis of photoaffinity -enriched datasets from Arabidopsis thaliana shoot samples. (a,b) V olcano plots of \nsignificantly enriched proteins (FDR < 0.05, S0 = 1) from competition experiments with free 4-PP-InsP5 (panel A) or free 6-\nPP-InsP5 (panel B) as competitors. Highlighted proteins (blue) were selected based on high enrichment scores and/or their \npotential relevance to inositol phosphate signaling and phosphoinositide metabolism, including: a) AT4G25050.1 (ACP4; acyl \ncarrier protein 4), AT1G65280.1 (DN AJ heat shock N -terminal domain-containing protein), AT2G44680.2 (CKB4; casein \nkinase II beta subunit 4), AT3G03790.1 (ankyrin repeat/RCC1 family protein), and AT5G65900.1 (DEA(D/H) -box RNA \nhelicase family protein).  b) AT1G80380.3 (P -loop containing nucleoside triphosphate hydrolases superfamily protein), \nAT2G28390.1 (SAND family protein), AT4G31160.1 (DCAF1; DDB1-CUL4 associated factor 1), AT1G54630.1 (ACP3; acyl \ncarrier protein 3), and AT3G05020.1 (ACP1; acyl carrier protein 1). c) UpSet plot illustrating the overlap of enriched proteins \nacross all shoot samples from photoaffinity enrichment experiments, including datasets from both custom and commercial \nlinker experiments with free 4-PP-InsP5 and 6-PP-InsP5 as competitors. d) Gene Ontology (GO) analysis of enriched proteins \nfrom shoot samples, showing Driver Terms identified by g:Profiler. For this analysis, protein lists from on -bead and elution \nfractions were combined. Categories are grouped into molecular function (blue) and biological process (red). \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 16 \nGO-term enrichment, analyzed separately for root and shoot samples, revealed pronounced \ntissue-specific differences. In roots, categories linked to RNA processing and transcription, \nprotein folding, and phosphatidylinositol metabolism were strongly enriche d. Shoots instead \nshowed enrichment of small -molecule metabolic processes, ion - and metabolite -binding \nactivities, and photosynthesis -related terms, consistent with their distinct physiological roles. \nNotably, the term “response to temperature stimulus” wa s enriched in root samples, while \n“response to cold” appeared in shoots, suggesting that 4/6‑PP‑InsP 5 (4/5) may contribute to \ntemperature-related stress responses. This observation complements recent findings implicating \n4/6‑PP‑InsP5 (4/5) in thermal signaling pathways in plants.[12] Collectively, these data indicate \n4/6-PP-InsP5 (4/5) may modulate diverse biological pathways, requiring detailed functional \nvalidation in follow-up studies. \nThese results further demonstrate the usefulness of photoaffinity-based approaches to reveal a \nbroad spectrum of potential 4/6 -PP-InsP5 interactors, while showing that probe design, \ncompetition strategy, and tissue context critically shape the captured interactome. Although the \ncustom linker provided higher sensitivity, it may also increase non-specific or transient binding, \nunderscoring the need for cautious interpretation and validation of candidate proteins. Together, \nthese findings offer a refined perspective on the application of photoaffinity strategies to dissect \ncomplex plant interactomes  of inositol pyrophosphates, potentially wit h information about \nisomer specific responses based on the competition isomer used. \n \nFigure 6: UpSet plots illustrating the overlap of enriched proteins across different enrichment strategies in Arabidopsis thaliana \nroot (panel a)  and shoot (panel b)  samples. Data include Affi -Gel, commercial, and custom photoaffinity enrichment \nexperiments. For Affi-Gel, on-bead and elution fractions were combined. For photoaffinity enrichment experiments, protein \nlists from both competition experiments were combined prior to analysis. \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 17 \nTogether, the Affi -Gel and photoaffinity enrichment strategies provided complementary \nperspectives on the Arabidopsis 4/6-PP-InsP5 interactome. The Affi-Gel method reproducibly \ncaptured a focused, tissue -independent subset of high -affinity interactors, while photoaffinity \nenrichment expanded coverage to include weaker and transient interactions stabilized through \ncovalent crosslinkin g. Both approaches consistently enriched proteins associated with \nphosphatidylinositol metabolism and inositol phosphate signaling. Across datasets, more \nproteins were displaced by 6 -PP-InsP5 (5) than by 4 -PP-InsP₅ ( 4), suggesting a broader \ninteractome and potentially a more pronounced signaling role for this isomer. This observation \naligns with prior findings that implicate 6 -PP-InsP5 (5) as the biologically relevant isomer in \nDictyostelium discoideum.[37] Yet, of course the situation in plants might be different and future \nstudies will have to provide clarity about the enantiomer identity of these PP-InsPs. \nSeveral proteins with established inositol phosphate - or lipid-binding domains, such as PH, \nSPX, or C2, were reproducibly detected across datasets  – including AT4G14740.2 (PH -like \ndomain), AT3G59660.1 (C2 domain), and AT1G35350.1 (EXS family; SPX-related) – further \nsupporting the specificity and reliability of the enrichment approaches , as these domains have \npreviously been implied in PP-InsP binding.[3,6] \nTo evaluate whether the enrichment approach yields proteins capable of direct ligand \ninteraction, we selected candidate interactors for biophysical validation based on favorable \nexpression characteristics. Specifically, we prioritized small, predicted soluble proteins and in \nparticular abundant domains lacking predicted transmembrane regions to facilitate recombinant \nproduction and downstream analysis. Among the considered candidates, AtFHA2 \n(AT3G07220.1), a FHA domain-containing protein, was the first to yield sufficient amounts of \nproperly folded protein and was therefore selected for follow -up experiments.  FHAs are \ndomains present in at least 18 genes encoded by the Arabidopsis genome and they also occur \nin other eukaryotes and eubacteria.[38] In our screen, it was identified in the root photoaffinity \npulldown using the commercial linker and 4 -PP-InsP5 (4) as competitor. Given its \nphosphothreonine-binding FHA domain, [39] AtFHA2 might be involved in phosphorylation -\ndependent signaling processes that intersect with inositol pyrophosphate pathways.  \nLigand binding was confirmed by isothermal titration calorimetry (ITC), which demonstrated \ndirect interaction of AtFHA2 with InsP6 (6) and 4-PP-InsP5 (4) (see Figure 7A and 7B). While \nInsP6 (6) showed only weak binding with a dissociation constant of ca. 34 µM, 4-PP-InsP5 (4) \nbound substantially more tightly, with a K d of ca. 4  µM. These results confirm a markedly \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 18 \nhigher binding affinity of 4 -PP-InsP5 (4) under the tested conditions.  These two ligands were \nselected based on their relevance to the experimental design: InsP 6 (6) served as a broadly \nestablished reference, while 4-PP-InsP5 (4) corresponded to the competition ligand used during \nthe enrichment. A broader comparison with additional PP -InsP5 isomers was not pursued, as \nprotein yield was limited and 4-PP-InsP5 (4) was directly linked to the dataset in which AtFHA2 \nwas identified. Circular dichroism (CD) spectroscopy further confirmed a well -folded \nsecondary structure and revealed minor conformational changes upon ligand addition in a dose-\ndependent manner  (see Figure 7C). Taken together, the direct in vitro  interaction between \nAtFHA2 and 4 -PP-InsP5 (4) exemplifies the ability of the enrichment strategy to uncover \nrelevant binding partners for further in vivo validation. \n \nFigure 7: Biophysical validation of AtFHA2 as a ligand-binding protein. A) ITC analysis of InsP6 (6) binding to recombinant \nAtFHA2: raw titration data and corresponding binding isotherm. B) ITC analysis of 4 -PP-InsP5 (4) binding to AtFHA2, \ndisplayed as raw data and fitted isotherm. C) Circular dichroism (CD) spectra of AtFHA2 in the absence and presence of InsP6, \nconfirming a folded structure and no major conformational changes upon ligand binding. \n3 Conclusion \nIn this study, we established complementary Affi -Gel- and photoaffinity -based enrichment \nstrategies to systematically map the protein interactome of 4/6 -PP-InsP5 (4/5) in Arabidopsis \nthaliana. Affi-Gel preferentially enriched a consistent core of high-affinity interactors, whereas \nphotoaffinity labeling, applied for the first time to inositol pyrophosphate pull-downs, revealed \na broader, context -dependent spectrum of proteins, reflecting both stable and transient \ninteractions. The custom linker (compound 20) outperformed the commercial reagent  \n(compound 21), demonstrating higher capture efficiency. Competition experiments further \nshowed that 6 -PP-InsP5 (5) displaced a more diverse set of proteins than 4 -PP-InsP5 (4), \nsuggesting isomer-specific roles in plant signaling. Functional annotation highlighted strong \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 October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683386doi: bioRxiv preprint \n\n 19 \nlinks to phosphatidylinositol metabolism and inositol phosphate signaling,  as well as \ntemperature stress related proteins. R ecombinant validation of AtFHA2 provided direct \nevidence for specific ligand binding.  Since multiple FHA domain-containing proteins exist in \nArabidopsis and other organisms, it might be established as another general PP -InsP binding \ndomain, much like SPX, PH, and C2. Together, these findings establish a versatile approach for \nPP-InsP interactome mapping in plants and provide a basis for future studies to clarify the \ncellular roles and signaling functions of this underexplored isomer. \nSupporting Information \nThe authors have cited additional references within the Supporting \nInformation.[11,14,16,24,27,31,32,34,35,40 –43] \nAcknowledgement \nWe thank Dr. Stefan Braukmüller and Dr. Manfred Keller from MagRes at the University of \nFreiburg for their support and for providing a significant amount of NMR measurement time. \nWe also thank Christoph Warth for HRMS measurements and Guizhen Lui, Mengsi Lu and \nIsabel Prucker for CE -MS measurements. We also thank Anne Harzen for performing in \nproteomics sample preparation  and Brigitte Ueberbach for technical assistance with \nArabidopsis cultivation and plant lysate preparation. This study was supported by the Deutsche \nForschungsgemeinschaft (DFG) (Project ID 560443421, JE 572/11-1, to H.J.J.; SCHA 1274/5-\n1, to G.S.) and under Germany’s Excellence Strategy (CIBSS, EXC -2189, Project ID \n390939984, to H.J.J.; PhenoRob, EXC-2070-390732324, to G.S.). H.J.J. acknowledges funding \nfrom the V olkswagen Foundation (VW Momentum Grant 98604). D.L. is grateful to the funding \nfrom Ministry of Education, MoE-STARS/STARS-2/2023-0162. ChatGPT (OpenAI, GPT‑4o) \nwas used for language refinement and support in generating R scripts f or data analysis and \nvisualization. The Table of Contents Figure and Figure 2 were created using BioRender.com. \nConflict of Interest \nThe authors declare no conflict of interest. \nLiterature \n[1] S. B. Shears, Adv. Biol. 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