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MatzingerMass SpectrometryChemical cross-linking coupled with mass spectrometry proteomicsNot availableCross-linkingAffinity enrichmentDbcoClick reactionDsbsoMass spectrometryhttp://www.ebi.ac.uk/pride/archive/projects/PXD016963Cross-linking of Cas9 or E. coli ribosome using DSBSO (1 mM, 1 h, room temperature). For ribosome samples, an additional denaturation step (Na-DOC, 1.5 %) and digestion of nucleotides using benzonase was performed. Reduction of proteins was perforemd using DTT (10 mM, 30 min, 56 ������C) and alkylation was done using IAA (20 mM, 30 min, room temperature, in the dark). Digestion was done sequentially using LysC (2 h, 37 ������C) and trypsin (16 h, 37 ������C) in a 1:50 (w/w) ratio each. Enrichment of resulting cross-linked peptides using DBCO or BARAC biotin method as described in the mansucript, folllowed by LC - MS/MS measurementPrideNot availableiodoacetamide derivatized residuemonohydroxylated residueMS data were analyzed with the help of Thermo Proteome Discoverer (2.3.0.523). Peptide identification was performed by MS Amanda (2.3.0.12368). The peptide mass tolerance was set to ������5 ppm and the fragment mass tolerance to ������0.02 Da. Carbamidomethyl (+57.021 Da) at cysteine was set as static modification. Oxidation (+15.995 Da) at methionine was set as dynamic modification. The result was filtered to 1% FDR (false discovery rate) on peptide level using the Target Decoy PSM Validator integrated in Thermo Proteome Discoverer, PSM hits were additionally filtered for a minimum score of 150. Cross-links were identified either using XlinkX 2.225 as node within Proteome Discoverer (2.3.0.523) or using MeroX (2.0.0.6) as indicated. For XlinkX the cross-link modification DSBSO was defined as C11H16O6S2 with the following crosslink-fragments: Alkene C3H2O, thiol C8H12O4S2, sulfenic acid C8H14O5S2 and linker specificity towards lysine and N-terminal amino residues was set. Fixed carbamidomethylation of cysteine and variable oxidation of methionine residues were set as modifications. Standard settings were used, with a minimal XlinkX score of 40 and a minimal delta score of 4 and results were filtered at 5 % FDR at peptide level using the XlinkX validator node. To analyze data with MeroX, raw files were first converted to mgf format using MSConvertGUI (3.0.19085-a306312d7) without using any filter. Data analysis was performed using the following settings: C-terminal cleavage sites lysine and arginine with 3 missed cleavages, allowed peptide length: 5 to 30, as static modification acetamidation of cysteine and as variable modification oxidation of methionine was set. The cross-linker DSBSO was defined as follows: DSBSO with specificity towards lysine and N-termini, fragments at site 1 and 2: Alkene and Thiol, as given for XlinkX above. Additionally, the following diagnostic ions were set: C8H12NO, C8H15N2O, C8H12NOS, C8H15N2OS. Dead ends were allowed to react with H2O. Further settings: precursor precision 4 ppm, fragment ion precision 8 ppm, S/N ratio 1.5, precursor masses were corrected (max 3 isotope shifts). Prescore intensity 10 %, FDR cutoff 5 %, score cutoff -1, for analysis with large databases, the proteome wide mode with a minimum peptide score of 10 was used, otherwise RISEUP mode was used. Quantification of cross linked peptides was performed label free using apQuant (3.1.1.27312)30 within Proteome Discoverer 2.3. As database for Cas9 samples the sequence of S. Pyogenes Cas9 with fused HaloTag������ plus the full human Swiss-Prot (as of 13th March 2018, 20271 proteins) was used. For ribosome samples a shotgun database containing 171 proteins, generated earlier9, was used and in experiments spiked with tryptic HEK peptides the full human Swiss-Prot (as of 13th March 2018, 20271 proteins) was addedProteomicsKarl MechtlerOrbitrap Fusion LumosQ Exactive HFPARTIALIMP, Campus Vienna Biocenter 1, 1030 Vienna, AustriaHomo Sapiens (human)Escherichia Coli32250121 Matzinger M, Kandioller W, Doppler P, Heiss EH, Mechtler K. Fast and Highly Efficient Affinity Enrichment of Azide-A-DSBSO Cross-Linked Peptides. J Proteome Res. 2020 19(5):2071-2079 10.1021/acs.jproteome.0c00003manuel.matzinger@imp.ac.atEpic-xsIMP / University of ViennaAustriaCross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to substochiometric reaction efficiencies, cross-linked peptides are usually low abundance. This results in challenging data evaluation and the need for an effective enrichment. Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides using dibenzocyclooctyne (DBCO) coupled Sepharose beads. We probed this method using recombinant Cas9 and <i>E. coli</i> ribosome. For Cas9, the number of detectable cross-links was increased from ∼100 before enrichment to 580 cross-links after enrichment. To mimic a cellular lysate, <i>E. coli</i> ribosome was spiked into a tryptic HEK background at a ratio of 1:2-1:100. The number of detectable unique cross-links was maintained high at ∼100. The estimated enrichment efficiency was improved by a factor of 4-5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 μg cross-linked <i>E. coli</i> ribosomes in a background of 100 μg tryptic HEK peptides, indicating a high enrichment sensitivity. In contrast to conventional enrichment techniques, like SEC, the time needed for preparation and MS measurement is significantly reduced. This robust, fast, and selective enrichment method for azide-tagged linkers will contribute to mapping protein-protein interactions, investigating protein architectures in more depth, and helping to understand complex biological processes.Fast and Highly Efficient Affinity Enrichment of Azide-A-DSBSO Cross-Linked Peptides.Matzinger Manuel M, Kandioller Wolfgang W, Doppler Philipp P, Heiss Elke H EH, Mechtler Karl Kprojections, Eph-like tyrosine kinase 1, vitamin B7, Sepharose C1 4B, ribosomal RNA, ETK1, protein, organ process., Strepavidin, Long Term, Productivity, peptide, Polypeptides, protein polypeptide chains, peptido, 5-[(3aS, Biotin, GRP1, Grp1, SEC, 1, 6aR)-Hexahydro-2-oxo-1H-thieno[3, Analysis, protein aggregate, Human embryo kinase, Effect, present in fewer numbers in organism, TYRO4, Mass Spectrum Analysis, (R*, Sepharose, increased, me75, peptides, Analyses, BAG-3, PTPSTEP, EK4, (3aS, sec, cis-Hexahydro-2-oxo-1H-thieno(3, biotina, hypoplasia, proteins, sulphoxide, biotine, number of, BIOTIN, Estimated, D17Mit170, T1, Fast, allergic reaction, reaction, C1 4B, decreased, Hermes, l(2)k08110, 6-anhydro-alpha-L-galactopyranosyl-(1->3)-beta-D-galactopyranan, papilla, sulfoxide, biotinum, Long-Term Effects, Azide, FAST, GPH, ratio, Medobiotin, anatomical protrusion, PLATEST, Rombellin, 4B, R*)-isomer, acid, lamina, Longterm Effect, flanges, FASTK, extra or missing physical or functional parts, Tl3, Tl2, Spectrum Analysis, results, 4S, 4-d)imidazole-4-pentanoic acid, (3aS-(3aalpha, Spectroscopy, malonyl-CoA:benzoyl-CoA malonyltransferase activity, [4)-3, 3.1.3.48, Sepharose C1, shelf, EPH-like kinase 4, Biotin Ratiopharm, 4-d)imidazoline-4-valeric acid, D-Biotin, Acid, 1H-Thieno(3, MFM6, Biotine Roche, Step, CG11628, acide, shelves, proportionality, Spectrometry, acids, rate, Striatum-enriched protein-tyrosine phosphatase, hEK4, acido, S*)-isomer, lysed material, projection, 2-(methylenedioxy)-4-(2-(octylsulfinyl)propyl)benzene, ridge, STEP, spine, BIS, Bis, hypersensitivity reaction disease, Sepharose 4B, HEK, cis-(+)-Tetrahydro-2-oxothieno[3, Polypeptide, Tyrosine-protein kinase receptor ETK1, D-(+)-biotin, Biokur, time, Platelets, accessory, 4-d]imidazol-4-yl)pentanoic acid, Deacura, GRP1/cytohesin 1, Effects, lamellae, Biotine, number, STK10, Medebiotin, Spectrum Analyses, Vitamin H, protein-containing complex, supernumerary, process of organ, protrusion, CG11633, cytohesin/GRP1, period, lamella, method, HEK4, polypeptide chain, reduced, sensitive, subnumerary, method used in an experiment, 4beta, Mass, Low, tiny, 6aalpha))-, Gelfert, sensitivity, Mass Spectroscopy, Tyrosine-protein kinase TYRO4, T6K12_14, study, proportion, Biotin Hermes, Coenzyme R, Longterm, Selb, l(2)SH2 0323, 2-1, ridges, Long-Term, decreased number, NSC-8400, 4-d]imidazole-4-valeric acid, sulfoxyl, lysate, 2.7.10.1, Neural-specific protein-tyrosine phosphatase, ATP:Fas-activated serine/threonine protein phosphotransferase activity, (+)-cis-Hexahydro-2-oxo-1H-thieno[3, piperonyl sulfoxide, mg638, Agarose, has or lacks parts of type, peptidos, hypersensitivity reaction, Long-Term Effect, Saeure, laminae, 6-An-alpha-L-Galp-(1->3)-beta-D-Galp-(1->]n, ETK, small, AA407278, data, cou, protein complex, anatomical process, Roche, stepk, Biotin Gelfert, isosafrole octyl sulfoxide, 5-(2-oxohexahydro-1H-thieno[3, Peptide, 4]imidazole-4-valeric acid, mereological quality, polypeptide, 6aR)-2-oxohexahydro-1H-thieno[3, Lr, cis-Tetrahydro-2-oxothieno(3, secret agent, 4]imidazoline-4-valeric acid, biotin, native protein, SPR-2A, natural protein, Protein, 4-d]imidazol-4-yl]pentanoic acid, Long Term Effects, background, membrane bound ribosome, l(2)SH0323, Mass Spectrum Analyses, flange, Biodermatin, free ribosome, T6K12.14, CYH1, Mass Spectrum, Biotin-Ratiopharm, Ribosome, hypersensitivity, hypersensitive, Gabunat, sulfox-cide, underdeveloped, Saeuren, increased number, hexahydro-2-oxo-, (1->4)-3, 4)imidazole-4-valeric acid, DmelCG11628, introduction, Longterm Effects, plan specification, CAIR-1, present in greater numbers in organism, processes, process, cardinality, Bra, quotient, processus, PeptidWater, Eph-like tyrosine kinase 1, dmMOF, Raw, ion, Lysine Hydrochloride, primitive node, Irip, ribosomal RNA, BOUND WATER, oxidane, NAALAdase, WATER, HOH, 5730420M11Rik, DmelCG9648, Polypeptides, NAALADase I, FOLH, Lysine Acetate, Drug Tolerance, dorsal marginal zone, dMOF, C530001K22Rik, 2, Analysis, myd, Human embryo kinase, TYRO4, WMS, L Lysine, somitogenic mesoderm, SET, D, (S)-2-amino-5-guanidinopentanoic acid, F, DELTA, Membrane glutamate carboxypeptidase, Analyses, dead, K, M, C1, phosphatase 2A inhibitor I2PP2A, EK4, R, Arginine, Mbp-1, Kitl, Monohydrate DL-Arginine Acetate, proteins, Y, Mast cell growth factor, DmelCG4299, DELTA1, set, LYS, Hensen node, Lysin, delta D1, fused to, Delta, Half Cystine, CG3619, drug tolerance, embryo organizer, SGS, DL, 1053/14, Zinc Cysteinate, unsegmented mesenchyme, immune system tolerance, somatomedin, arginine, IBP1, SH2-B PH domain-containing signaling mediator 1, ACMICD, bHLHd7, bHLHd6, bHLHd9, bHLHd8, H2O, bHLHd5, bHLHd4, DL1, NF-E1, mKIAA0609, Methionin, methionine, Hmet, fg, HLA-DR-associated protein II, DI-2, I-2Dm, FGCP, Stem cell factor, FU, proportionality, expanded, rate, alpha, inhibitor of granzyme A-activated DNase, 1304/03, STAT5, CT12133, human, I-2PP1, Pro-rich, stem cell factor, MDC1D, Self Tolerance, TAF-IBETA, enr, Fu, DMZ, 2018, TAF-Ibeta, l(3)92Ab, fused, Liquimeth, Gb, big, FUSED, l(3)j8C3, knobbly, 2-amino-4-(methylsulfanyl)butanoic acid, Clo, False, region or site annotation, Slf, SLF, Fused, precursor, agua, large, DmelCG3025, Gene Products, Lysine, Half-Cystine, shield, proportion, positional, MAX, nodus primitivus, L Cysteine, MDDGB6, INO80S, 2pp2a, Spemann's organizer, ions, man, CG10574, Con, max, 2PP2A, 2-amino-4-(methylthio)butanoic acid, dSET, dSet, blz, L-Arg, peptidos, Kb, metionina, ETK, Ki, hematopoietic growth factor KL, 1440/11, Proteins, CG3025, L-Isomer, embryonic organizer, B930045J24, polypeptide, Immune Tolerance, I-2PP2A, L-Isomer Arginine, Acetate, Dm I-2, 0495/20, chemical analysis, post-mortem, MFS1, segmental plate, Sh2bpsm1, Death, L-Lysine, Met, free ribosome, Ribosome, Col4a-1, Protein., (2S)-2-amino-5-guanidinopentanoic acid, primary structure of sequence macromolecule, Folate hydrolase 1, Gene Proteins, 1485/04, binding_or_interaction_site, MGF, Mgf, quotient, embryonic shield, 225, [OH2], Enisyl, Data Analyses, Immunological Tolerance, SH2-Bb, deceased, IPP2A2, Bru, delta, determination, L-arginine, DL-Arginine Acetate, Somatomedin-C, cleavage, Mbp1, Monohydrate, ETK1, Cardiac Death, contrasted, UCRBP, binding site, peptide, YIN-YANG-1, L-Isomer Methionine, Methionine, ClvPrd, peptido, 0926/11, kinky, node, 3.4.17.21, Arg, SH2-B, Svc, peptides, TAF-I, dMax, (S)-2-Amino-5-guanidinovaleric acid, somatomedin-C, epsilon-diaminocaproic acid, SF, NAALAD1, free, Folylpoly-gamma-glutamate carboxypeptidase, IGAAD, DmelCG10574, IGF1, ppm, AI316800, Mechano growth factor, Hensen's node, dihydridooxygen, Soluble KIT ligand, dihydrogen oxide, GPHYSD2, Sl, Racemethionine, ratio, L-Arginin, phapii, IGF-I, steel factor, wide/broad, gyltl1b-b, T5E21.12, 2-Amino-4-(methylthio)butyric acid, Arginine Hydrochloride, aqua, CG9648, StF-IT-1, results, sKITLG, Ionen, PSM, Psm, MDDGA6, mechano growth factor, dmax, lysine, EPH-like kinase 4, Immunologic Tolerance, Henson's node, presumptive somite mesoderm, Prostate-specific membrane antigen, Igf-1, fixed, region, T5E21_12, L-Arginine, parent ion, SHEP7, Pteroylpoly-gamma-glutamate carboxypeptidase, gyltl1b, positional polypeptide feature, mast cell growth factor, DmelCG3619, mdc1d, hydrogen hydroxide, L-Cysteine, hEK4, MASS, CG4299, merged with, acqua, Kitlg, wide, MOF, Mof, KL-1, Tolerance, precursor ion, enlarged, HEK, PSMA, KITLG, Polypeptide, Wasser, Tyrosine-protein kinase receptor ETK1, i2pp2a, Proteomes, SH2 domain-containing protein 1B, Cysteine Hydrochloride, human being, anon-WO0118547.269, unsegmented paraxial mesoderm, L Isomer, FBN, Gene, FPH2, broad, somitomeric mesoderm, GCPII, PHAPII, froggy, Gyltl1a, L-(+)-arginine, HEK4, template-activating factor I, (2S)-2-amino-5-(carbamimidamido)pentanoic acid, ECTOL1, L-Methionine, N-acetylated-alpha-linked acidic dipeptidase I, mKIAA1299, Del(8)44H, Pedameth, Tyrosine-protein kinase TYRO4, SH2B, 1119/09, iones, ipp2a2, mGCP, eau, OCTD, BPFD#36, 2.7.10.1, 10^[-6], LMPY, taf-ibeta, stem node, great, dmDelta, site, mKIAA1278, organizer, data, l(3)05151, DL-Methionine, igaad, total expressed protein, DL Arginine Acetate, Peptide, GCP2, AI875693, Glutamate carboxypeptidase II, Axin, Cell growth-inhibiting gene 27 protein, water, Protein, I2PP2A, Spemann Mangold organizer, Hydrochloride, sequence, L Arginine, membrane bound ribosome, WMS2, Data Base, PH and SH2 domain-containing signaling mediator, nodal stem, INSDC_feature:misc_binding, 1423/11, E(ls)2, Protein Gene Products, dSET/TAF-Ibeta, 2610030F17Rik, Ion, Data, joined with, SSKS, AI425885, c-Kit ligand, AA960152, alpha-amino-gamma-methylmercaptobutyric acid, Peptid, assay, SCF, variable, AA407739, 6-diaminohexanoic acid, 1700112N14Rik, coalescedpolypeptide, peptide, Polypeptides, peptido, peptides, ATP:Fas-activated serine/threonine protein phosphotransferase activity, STK10, Peptid, peptidos, Polypeptide, FASTK, Peptide., FAST, Azide, Fastpolypeptide, peptide, Polypeptides, peptido, peptides, ATP:Fas-activated serine/threonine protein phosphotransferase activity, STK10, Peptid, peptidos, Polypeptide, FASTK, Peptide., FAST, Azide, FastHac-1/Dark, 4-d]imidazol-4-yl)pentanoic acid, Deacura, GRP1/cytohesin 1, Dapaf-1/HAC-1, vitamin B7, DmelCG6829, Biotine, Medebiotin, ribosomal RNA, Gene, 2-(indol-3-yl)ethanoic acid, Vitamin H, Apaf-1, CG7826, CG11633, Alkylations, Dark/Hac-1/dApaf1, cytohesin/GRP1, peptide, Polypeptides, method, Hac1, Dark/Hac-1/dApaf-1, IAA, peptido, 5-[(3aS, Biotin, GRP1, Grp1, 4beta, Gene Products, CG7835, CG42273, Min, 6aR)-Hexahydro-2-oxo-1H-thieno[3, Apaf1, 6aalpha))-, ARK, Gelfert, DmelCG42273, proportion, Biotin Hermes, hac-1, peptides, Coenzyme R, PTPSTEP, weight-weight percentage, (3aS, dapaf-1, beta-Trypsin, cis-Hexahydro-2-oxo-1H-thieno(3, biotina, l(2)SH2 0323, min, arc, mAPC, proteins, Indoleacetic acid, dapaf, biotine, dark, BIOTIN, ark, T1, AI047805, 4-d]imidazole-4-valeric acid, Neural-specific protein-tyrosine phosphatase, Hermes, (indol-3-yl)acetic acid, method used in an experiment., DARK, (+)-cis-Hexahydro-2-oxo-1H-thieno[3, l(2)k08110, peptidos, dApaf1, biotinum, Nucleotide, dArk, Dapaf-1, GPH, ratio, 1H-indol-3-ylacetic acid, Medobiotin, dark/dapaf-1/hac-1, DYRK1, mass percentage, D-Apaf-1, Rombellin, dApaf-1, Proteins, anon-53Fa, Dmel_CG7826, l(2)SH0173, Roche, stepk, Biotin Gelfert, apaf1, 5-(2-oxohexahydro-1H-thieno[3, Peptide, 4]imidazole-4-valeric acid, 4S, 4-d)imidazole-4-pentanoic acid, (3aS-(3aalpha, polypeptide, 6aR)-2-oxohexahydro-1H-thieno[3, APAF1, (Indol-3-yl)acetate, cis-Tetrahydro-2-oxothieno(3, 4]imidazoline-4-valeric acid, biotin, Temperatures, Dm1, Hac-1, Protein, 4-d]imidazol-4-yl]pentanoic acid, 3.1.3.48, Biotin Ratiopharm, Dyrk1, IES, Dark, 4-d)imidazoline-4-valeric acid, membrane bound ribosome, l(2)SH0323, Dark/Apaf-I, CG6829, Dmel_CG7835, D-Biotin, Biodermatin, free ribosome, dark/hac-1/dapaf-1, CYH1, Mnb, MNB, AU020952, Biotin-Ratiopharm, 1H-Thieno(3, Ribosome, Biotine Roche, Gabunat, Step, CG11628, CC1, proportionality, 3-Indolylessigsaeure, Tripcellim, hexahydro-2-oxo-, Dark/Dapaf-1/HAC1, rate, Striatum-enriched protein-tyrosine phosphatase, hac1, AW124434, 4)imidazole-4-valeric acid, beta Trypsin, dApaf-1/DARK/HAC-1, DmelCG11628, w/w, ME-IV, Protein Gene Products, plan specification, dapaf-1S, Gene Proteins, Trypure, Indole-3-acetic acid, apaf-1, dapaf-1L, heteroauxin, STEP, quotient, cis-(+)-Tetrahydro-2-oxothieno[3, Peptid, Polypeptide, D-(+)-biotin, Biokur, dAPAF-1projections, Eph-like tyrosine kinase 1, type 2, vitamin B7, methionine aminopeptidase activity, ribosomal RNA, ETK1, protein, organ process., Strepavidin, Long Term, Productivity, peptide, Polypeptides, protein polypeptide chains, peptido, 5-[(3aS, Biotin, GRP1, Grp1, SEC, 1, 2, 6aR)-Hexahydro-2-oxo-1H-thieno[3, Analysis, protein aggregate, Human embryo kinase, Effect, present in fewer numbers in organism, TYRO4, peptidase M activity, Mass Spectrum Analysis, (R*, adenomas, increased, rabGAPLP, me75, L-methionine aminopeptidase activity, peptides, Analyses, BAG-3, PTPSTEP, EK4, (3aS, sec, cis-Hexahydro-2-oxo-1H-thieno(3, biotina, hypoplasia, RabGAP-5, proteins, sulphoxide, biotine, number of, BIOTIN, Estimated, D17Mit170, T1, Fast, allergic reaction, reaction, decreased, Hermes, RUSC3, l(2)k08110, papilla, sulfoxide, biotinum, Long-Term Effects, Azide, FAST, GPH, ratio, Medobiotin, anatomical protrusion, PLATEST, Rombellin, R*)-isomer, acid, MYH-associated polyposis, lamina, Longterm Effect, autosomal recessive familial adenomatous polyposis, Maps, flanges, FASTK, extra or missing physical or functional parts, Tl3, Tl2, Spectrum Analysis, results, 4S, 4-d)imidazole-4-pentanoic acid, (3aS-(3aalpha, Spectroscopy, malonyl-CoA:benzoyl-CoA malonyltransferase activity, 3.1.3.48, shelf, EPH-like kinase 4, Biotin Ratiopharm, 4-d)imidazoline-4-valeric acid, D-Biotin, autosomal recessive, MUTYH-associated polyposis, Acid, 1H-Thieno(3, MUTYH-related AFAP, RUTBC3, MFM6, Biotine Roche, Step, CG11628, acide, shelves, proportionality, Spectrometry, acids, rate, Striatum-enriched protein-tyrosine phosphatase, hEK4, acido, S*)-isomer, lysed material, projection, 2-(methylenedioxy)-4-(2-(octylsulfinyl)propyl)benzene, ridge, RABGAP5, STEP, spine, BIS, Bis, hypersensitivity reaction disease, multiple colorectal, HEK, cis-(+)-Tetrahydro-2-oxothieno[3, MUTYH-related attenuated familial polyposis coli, Polypeptide, Tyrosine-protein kinase receptor ETK1, D-(+)-biotin, Biokur, time, Platelets, accessory, 4-d]imidazol-4-yl)pentanoic acid, Deacura, GRP1/cytohesin 1, Effects, lamellae, Biotine, number, STK10, Medebiotin, Spectrum Analyses, Vitamin H, protein-containing complex, supernumerary, process of organ, protrusion, CG11633, cytohesin/GRP1, period, lamella, method, HEK4, polypeptide chain, reduced, sensitive, subnumerary, method used in an experiment, 4beta, Mass, Low, tiny, 6aalpha))-, Gelfert, sensitivity, familial adenomatous polyposis, Mass Spectroscopy, MAP, Tyrosine-protein kinase TYRO4, T6K12_14, study, proportion, Biotin Hermes, Coenzyme R, Longterm, familial adenomatous polyposis 2, Selb, l(2)SH2 0323, ridges, Long-Term, decreased number, autosomal recessive multiple colorectal adenomas, NSC-8400, 4-d]imidazole-4-valeric acid, sulfoxyl, lysate, 2.7.10.1, Neural-specific protein-tyrosine phosphatase, ATP:Fas-activated serine/threonine protein phosphotransferase activity, (+)-cis-Hexahydro-2-oxo-1H-thieno[3, piperonyl sulfoxide, mg638, has or lacks parts of type, peptidos, hypersensitivity reaction, Long-Term Effect, Saeure, laminae, ETK, small, AA407278, MUTYH-related attenuated familial adenomatous polyposis, data, cou, MUTYH-Associated Polyposis, protein complex, anatomical process, Roche, stepk, Biotin Gelfert, isosafrole octyl sulfoxide, 5-(2-oxohexahydro-1H-thieno[3, Peptide, 4]imidazole-4-valeric acid, mereological quality, polypeptide, 6aR)-2-oxohexahydro-1H-thieno[3, Lr, cis-Tetrahydro-2-oxothieno(3, secret agent, 4]imidazoline-4-valeric acid, biotin, native protein, natural protein, MUTYH-related attenuated FAP, Protein, 4-d]imidazol-4-yl]pentanoic acid, Long Term Effects, background, membrane bound ribosome, l(2)SH0323, Mass Spectrum Analyses, flange, Biodermatin, free ribosome, T6K12.14, CYH1, Mass Spectrum, Biotin-Ratiopharm, Ribosome, hypersensitivity, hypersensitive, Gabunat, sulfox-cide, underdeveloped, Saeuren, increased number, hexahydro-2-oxo-, 4)imidazole-4-valeric acid, MYH-Associated Polyposis, DmelCG11628, introduction, Longterm Effects, plan specification, CAIR-1, present in greater numbers in organism, processes, process, cardinality, Bra, quotient, processus, Peptid, MAP syndrome, FAP2, colorectal adenomatous polyposisfalseFast and highly efficient affinity enrichment of Azide-A-DSBSO cross-linked peptides.Cross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to sub-stochiometric reaction efficiencies, cross-linked peptides are usually low abundant. This results in challenging data evaluation and the need for an effective enrichment. Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides using dibenzocyclooctyne (DBCO) coupled Sepharose������ beads. We probed this method using recombinant Cas9 and E. coli ribosome. For Cas9, the number of detectable cross-links was increased from ~100 before enrichment to 580 cross-links after enrichment. To mimic a cellular lysate, E. coli ribosome was spiked into a tryptic HEK background at a ratio of 1:2 ��������� 1:100. The number of detectable unique cross-links maintained high at ~100. The estimated enrichment efficiency was improved by factor 4 -5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 ������g cross-linked E. coli ribosome in a background of 100 ������g tryptic HEK peptides, indicating a high enrichment sensitivity. In contrast to conventional enrichment techniques, like SEC, the time needed for preparation and MS measurement is significantly reduced. This robust, fast and selective enrichment method for azide-tagged linkers will contribute to map protein-protein interactions, investigate protein architectures in more depth and help to understand complex biological processes.2020-05-112020-02-24PXD016963445441009064152499175405499865931173880433303878343235983906327160884019373349615198215275838575748782721513517505898507655571480468353649215891030641898536413885623980332715910022619023888691564546374636013558811345061464796928145943280385833NCBITaxon:61579778118259040816329572633803945264127256314595361235178616722724323074699286745131123869408169NCBITaxon:245309544627912924970361251801424507284812355512744324264281504751029971438865990346941769453475154155405972936329979662877323962891575464784224295486208964317447127057558462821143193382352287230741114817362314896935293725126469039946727360106580240884204399472165999685835521659554440429412826071012832965431280339523218184318329204NCBITaxon:6191840235755478793128118365759307972185579190802143213870382426191692259106592226155153111795726070461832607052607078853187029455862652817363094317991036989361604882092855664678251249668425286192126385410943433175131971928753555432046NCBITaxon:10359NCBITaxon:13134565860688607699389422829795545351126806379599598704483006418022410023544326420367631076224308185431575412983964271116996328104215745771216979123899342321356223347472697049NCBITaxon:1773803010239297601011738185833424090699131274414163917018736745101165691569313076749409833422432640817212816120425468761383708127442399011912744201274426992551953883949113702100058999403430531156554285321895177311935014219328726317145051187236669872464268727492412153239564818822954767091115104580023055148015418006690313847562360314231361685962982510151098231174673493211676275910807726776733264837315344685NCBITaxon:961529722393765109767757357845849376083334191107927628086363266689NCBITaxon:2697049833321031298382375612413689975961248479749200360094319752283196627766896059606157295137221171101515151438992256737322501210000-0002-9765-7951