Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

Abstract

Ion mobility separates molecules in the gas-phase based on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a TOF mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking MS (XL-MS). Cross-linking reagents covalently link amino acids in proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent – termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Å2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50-70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides. Ion mobility separates molecules in the gas-phase based on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a TOF mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking MS (XL-MS). Cross-linking reagents covalently link amino acids in proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent – termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Å2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50-70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides. The folding of proteins, resulting in structural features that enable them to function and form complexes with other proteins, is one of the major driving forces in highly sophisticated cellular behavior. Misfolding and/or gain or loss of interactions to other proteins can lead to major dysfunction and potentially severe diseases (1Hartl F.U. Protein misfolding diseases.Annu. Rev. Biochem. 2017; 86: 21-26Crossref PubMed Scopus (160) Google Scholar, 2Lage K. Protein-protein interactions and genetic diseases: The interactome.Biochim. Biophys. Acta. 2014; 1842: 1971-1980Crossref PubMed Scopus (59) Google Scholar). Intimate knowledge of the structural details behind protein structures and interactions is of the utmost importance to develop novel treatments to interfere with these dysfunctions. Even though the study of protein structure is dominated by techniques like NMR, crystallography and cryo-EM, structural proteomics techniques driven by MS have an increasingly important, integrative role to uncover new details not achievable by the conventional techniques. For example, information on proteoforms (i.e. protein sequences and post-translational modifications) are typically not apparent with a technique like cryo-EM but are accessible by structural proteomics (3Chen B. Brown K.A. Lin Z. Ge Y. Top-down proteomics: ready for prime time?.Anal. Chem. 2018; 90: 110-127Crossref PubMed Scopus (83) Google Scholar). At the same time, spatial information within and between proteins can be obtained using cross-linking MS (XL-MS) (4Steigenberger B. Albanese P. Heck A.J.R. Scheltema R.A. To cleave or not to cleave in XL-MS?.J. Am. Soc. Mass Spectrom. 2020; 31: 196-206Crossref PubMed Scopus (21) Google Scholar, 5Iacobucci C. Piotrowski C. Aebersold R. Amaral B.C. Andrews P. Bernfur K. Borchers C. Brodie N.I. Bruce J.E. Cao Y. Chaignepain S. Chavez J.D. Claverol S. Cox J. Davis T. Degliesposti G. Dong M.-Q. Edinger N. Emanuelsson C. Gay M. Götze M. Gomes-Neto F. Gozzo F.C. Gutierrez C. Haupt C. Heck A.J.R. Herzog F. Huang L. Hoopmann M.R. Kalisman N. Klykov O. Kukačka Z. Liu F. MacCoss M.J. Mechtler K. Mesika R. Moritz R.L. Nagaraj N. Nesati V. Neves-Ferreira A.G.C. Ninnis R. Novák P. O'Reilly F.J. Pelzing M. Petrotchenko E. Piersimoni L. Plasencia M. Pukala T. Rand K.D. Rappsilber J. Reichmann D. Sailer C. Sarnowski C.P. Scheltema R.A. Schmidt C. Schriemer D.C. Shi Y. Skehel J.M. Slavin M. Sobott F. Solis-Mezarino V. Stephanowitz H. Stengel F. Stieger C.E. Trabjerg E. Trnka M. Vilaseca M. Viner R. Xiang Y. Yilmaz S. Zelter A. Ziemianowicz D. Leitner A. Sinz A. First community-wide, comparative cross-linking mass spectrometry study.Anal. Chem. 2019; 91: 6953-6961Crossref PubMed Scopus (43) Google Scholar, 6O'Reilly F.J. Rappsilber J. Cross-linking mass spectrometry: methods and applications in structural, molecular and systems biology.Nat. Struct. Mol. Biol. 2018; 25: 1000-1008Crossref PubMed Scopus (82) Google Scholar, 7Petrotchenko E.V. Borchers C.H. Crosslinking combined with mass spectrometry for structural proteomics.Mass Spectrom. Rev. 2010; 29: 862-876Crossref PubMed Scopus (147) Google Scholar, 8Liu F. Rijkers D.T.S. Post H. Heck A.J.R. Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry.Nat. Methods. 2015; 12: 1179-1184Crossref PubMed Scopus (252) Google Scholar). XL-MS typically uses small homobi-functional chemical reagents that irreversibly connect amino acids in close structural proximity. Most commonly highly reactive NHS-esters, which primarily capture the sidechains of lysines are used for this purpose. After reduction, alkylation and proteolytic digestion of the cross-linked proteins, three different products are formed: unmodified peptides, peptides with a quenched linker attached termed “mono-link” peptides and the desirable two peptides covalently connected by the cross-linking reagent termed “cross-link” peptides. Cross-linked peptides provide information on protein tertiary structure in the form of intra-links (two peptides from the same protein) and protein quaternary structure in the form of inter-links (two peptides from different proteins). As the reaction efficiency for cross-linking is estimated to be about 1-5%, and relatively few lysine pairs are found to be in sufficiently close proximity to be cross-linked, only 0.1% of the sample actually consists of cross-linked peptides, which substantially hampers their detection (9Steigenberger B. Pieters R.J. Heck A.J.R. Scheltema R.A. PhoX: An IMAC-Enrichable Cross-Linking Reagent.ACS Cent. Sci. 2019; 5: 1514-1522Crossref PubMed Scopus (37) Google Scholar, 10Chavez J.D. Bruce J.E. Chemical cross-linking with mass spectrometry: a tool for systems structural biology.Curr. Opin. Chem. Biol. 2019; 48: 8-18Crossref PubMed Scopus (57) Google Scholar, 11Leitner A. Walzthoeni T. Kahraman A. Herzog F. Rinner O. Beck M. Aebersold R. Probing Native Protein Structures by Chemical Cross-linking, Mass Spectrometry, and Bioinformatics.Mol. Cell. Proteomics. 2010; 9: 1634-1649Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). To focus the analysis, extensive pre-fractionation of the peptide mixture is commonly employed prior to the LC–MS measurement(s), using chromatographic techniques such as strong cation exchange (SCX) or size exclusion chromatography (SEC). However, reagents with an enrichment handle directly attached have emerged capable of removing the high background of normal peptides and uniquely enrich for modified peptide products (mono-linked and cross-linked peptides). For this purpose, conventionally a biotin handle is used, either directly attached to the reagent or introduced after the cross-linking reaction by a click-reaction. One of the downsides of using biotin as enrichment handle is that its high affinity binding to streptavidin prevents efficient elution from the enrichment beads. Recently, we developed and introduced a novel enrichable cross-linking reagent, PhoX, which is decorated with a phosphonic acid moiety directly attached on the cross-linking reagent (9Steigenberger B. Pieters R.J. Heck A.J.R. Scheltema R.A. PhoX: An IMAC-Enrichable Cross-Linking Reagent.ACS Cent. Sci. 2019; 5: 1514-1522Crossref PubMed Scopus (37) Google Scholar). This moiety is a stable mimic of a phosphate group and can therefore efficiently be enriched by IMAC-based techniques originally developed for phosphorylated peptides. Competing molecules for the affinity enrichment, such as phospho-peptides and nucleic acids, can selectively be removed by using phosphatase and/or benzonase, as PhoX remains stable under these conditions. With the PhoX enrichment handle, we increased the enrichment efficiency by up to 300× with 97% specificity, leading to excellent cross-link identification. The approach is however not yet focusing solely on the desired cross-linked peptides, as the sample still contains approximately 60% of the less informative mono-linked peptides. With ion mobility MS (IMMS) ions are separated over a time-frame of 10–100 ms by their collisional cross-section (CCS, Ω) (12Eiceman G. Karpas Z. Hill Jr, H. Ion mobility spectrometry. 3rd Ed. Taylor & Francis, UK2013Crossref Google Scholar, 13Uetrecht C. Rose R. van Duijn E. Lorenzen K. Heck A.J.R. Ion mobility mass spectrometry of proteins and protein assemblies.Chem. Soc. Rev. 2010; 39: 1633-1655Crossref PubMed Scopus (331) Google Scholar), which is based on their size, shape, and charge. Ion mobility separation (IMS) devices are typically installed between the liquid chromatography (LC) system and the mass analyzer. It has that ions from an can efficiently be with TOF as these devices have the high the of for this separation of are in the of with trapped ion mobility separation desirable properties, such as small size, low and highly efficient ion In ions are in an a allowing ions to be trapped and at different in the ion After ions can be from the by the and can be to a mass analyzer. mobility ions with CCS are from the by high mobility ions with CCS F. Beck S. N. M. O. M. sequencing and by in a trapped ion mobility 2015; PubMed Scopus Google Scholar, F. S. H. M. M. N. J. T. N. O. Cox J. O. M. with a novel trapped ion mobility mass Cell. Proteomics. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). As cross-linked peptides of two peptides connected by the cross-linking reagent, their size and typically from and mono-linked peptides and therefore we that the connected to a TOF be an excellent for the of separation Here, we the of XL-MS on the timsTOF Pro using the efficiently enrichable PhoX (9Steigenberger B. Pieters R.J. Heck A.J.R. Scheltema R.A. PhoX: An IMAC-Enrichable Cross-Linking Reagent.ACS Cent. Sci. 2019; 5: 1514-1522Crossref PubMed Scopus (37) Google Scholar). of the that the system has the to and the typically to cross-linked peptide with its to high The separates the mono-linked and cross-linked peptides, providing an of we a novel termed which of CCS information to an between molecules of and demonstrate the on protein mixture and a complex sample of proteins from a cellular A of the cross-linking reagent PhoX as (9Steigenberger B. Pieters R.J. Heck A.J.R. Scheltema R.A. PhoX: An IMAC-Enrichable Cross-Linking Reagent.ACS Cent. Sci. 2019; 5: 1514-1522Crossref PubMed Scopus (37) Google and at a of in This in and in at for as the reactive of PhoX can potentially to an to is to in the PhoX to peptides in at a of 2 The cross-linking reaction for one at and by of After with the peptide mixture directly the timsTOF with to cross-linked or mono-linked peptides and to with PhoX for at The cross-linking reaction quenched by of to a of cross-linking reagent removed by with three of or by Cross-linked proteins with of 2 for at by alkylation with of for at This reaction quenched by of of 2 the sample by with a of to protein) and to protein) for at after which acid to the digestion. peptides by prior to The for the different are as (1Hartl F.U. Protein misfolding diseases.Annu. Rev. Biochem. 2017; 86: 21-26Crossref PubMed Scopus (160) Google in with of K. Protein-protein interactions and genetic diseases: The interactome.Biochim. Biophys. Acta. 2014; 1842: 1971-1980Crossref PubMed Scopus (59) Google Protein of in with of (3Chen B. Brown K.A. Lin Z. Ge Y. Top-down proteomics: ready for prime time?.Anal. Chem. 2018; 90: 110-127Crossref PubMed Scopus (83) Google A cell of in benzonase, and by to a cell removed at for at The with of PhoX for at at a of by with of 2 for at and alkylation with of for at This reaction quenched by of of 2 the sample with and by with to protein) and to protein) for at after which acid to the digestion. peptides by prior to both enrichment as as LC–MS of cell lysate peptides as peptides at a of in A of of and the mixture at with using Cross-linked peptides enriched with as H. R. Heck A.J.R. and enrichment for low sample to 2017; PubMed Scopus Google Scholar), at a of with of and at a of with of The a in of and at a of the with of at a of and cross-linked peptides with of directly of and at to analysis, the in acid and approximately of peptides on the LC–MS either directly a or separated by on a with at a of A and with 0.1% acid and with acid 0.1% directly on the at a of In the at for increased to over the by an increase from to over For increased to for a and at that for an by to A. For experiments at different the between 2 and modified on the timsTOF Pro using from the for the for to The for mobility collision to at an mobility of and at collision between these two and or these and for of two at and of the collision To increase we the precursor to For based on collisional cross-section and monoisotopic mass of and a modified used that precursor CCS monoisotopic mass and a as between this is (i.e. the and this is The from with 2 from the and in with the developed tool for at The consists of two (1Hartl F.U. Protein misfolding diseases.Annu. Rev. Biochem. 2017; 86: 21-26Crossref PubMed Scopus (160) Google In the of the same precursor are combined a of the is with the precursor and mobility For the is estimated as the of the of in the with the is at of the is by over within of other a with the of the combined and the of K. Protein-protein interactions and genetic diseases: The interactome.Biochim. Biophys. Acta. 2014; 1842: 1971-1980Crossref PubMed Scopus (59) Google In the combined is are to a at of of of from resolved for Am. Soc. Mass Spectrom. PubMed Scopus Google Scholar), for of at and at with the an is information on the and monoisotopic The CCS are to the of in are of and the molecular of The for the peptides with using the same as to The for the experiments with O. B. S. D. Heck A.J.R. Scheltema R.A. and for mass spectrometry.Nat. 2018; PubMed Scopus Google Scholar). a the proteins under with a of commonly detected For the the proteins from a For peptides, a using protein by using mass spectrometry PubMed Scopus Google Scholar). as and protein as For the of mono-linked peptides, and as as the with a peptide of and up to two at at the peptide L. J.D. J. MacCoss M.J. for peptide from proteomics Methods. PubMed Scopus Google Scholar). For cross-linked peptides, a with PhoX as the cross-link as a and and protein as as digestion and up to two only with a of and a of at at the peptide a of the the protein are to and the cross-linked peptides are on protein analysis and of the with the and R. R. A for and 5: using H. for for with and as at at either peptide or protein or a As cross-linked peptides are different from unmodified peptides potentially not and we to the for the of cross-linked peptides Pieters R.J. Scheltema R.A. and using ions in collision peptide J. Mass Spectrom. 2019; Scopus Google Scholar, F. P. Scheltema R.A. Viner R. Heck A.J.R. and analysis for cross-link 2017; Scopus Google Scholar). To the collision for cross-linked peptide pairs on the timsTOF we directly cross-linked peptides, and the ions at both 2 as as to from 10–100 in of After the we on the of as as the of a cross-link ion from the lysine of peptide connected the to the peptide Pieters R.J. Scheltema R.A. and using ions in collision peptide J. Mass Spectrom. 2019; Scopus Google Scholar). The from this analysis for the cross-linked peptide at and for the cross-linked peptide at and the collision based on the and to the in This the collision to as this typically is observed for peptides, and the collision to as at this typically to be observed for peptides. 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J. Y. R. H. the of in peptide using 9: PubMed Scopus Google Scholar). the of the two different that close to the same of cross-linked peptides as the the of by of the sequences an of to between the using the within the A of found in the the which the which can be to in the CCS from the that can in by a of potentially driving the the A total of originally detected the not in the which can be by the same in detected CCS by in the mass detection are not cross-linked peptides the an increase of that by focusing the to a of of can be of to a PhoX enriched cross-linked cellular lysate that separation of the different classes of peptides is and not in the high To approach still at this of we the of the of cross-linked and peptides, and found to observed in the and the protein between the and cross-link of with the same as used a of the the of the two different that cross-linked peptides as the the of by of the sequences of the an of between the the within the as observed for the of the found of the and found of the with the However, a high of cross-link be the by the of focusing cross-link using CCS and monoisotopic mass the for cross-linking is to approximately of the the sequencing efficiency that is in both the as as the In the of a at high of the of a that in and be the to this and the of cross-linked peptides. to the MS in and therefore For the background not the cross-link in the of cross-link This the of within the for which we observed of precursor is employed to sequencing of the background of unwanted classes of For this purpose, with a of are in the of the focusing the mass to cross-linked peptides. for the such a is with the introduced provides an in for from to from to from to of the cross-link from to from to from to The of increasingly for the high potentially the we observed for these the at the of and a For the is less at sequencing from to from to from to is however in loss of cross-link from to from to from to at are typically and to informative can uniquely structural and that still provides an for from to from to from to its to from to from to from to XL-MS a approach to uncover structural details of proteins and J.D. Bruce J.E. Chemical cross-linking with mass spectrometry: a tool for systems structural biology.Curr. Opin. Chem. Biol. 2019; 48: 8-18Crossref PubMed Scopus (57) Google Scholar, 11Leitner A. Walzthoeni T. Kahraman A. Herzog F. Rinner O. Beck M. Aebersold R. Probing Native Protein Structures by Chemical Cross-linking, Mass Spectrometry, and Bioinformatics.Mol. Cell. Proteomics. 2010; 9: 1634-1649Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar), in highly complex (9Steigenberger B. Pieters R.J. Heck A.J.R. Scheltema R.A. PhoX: An IMAC-Enrichable Cross-Linking Reagent.ACS Cent. Sci. 2019; 5: 1514-1522Crossref PubMed Scopus (37) Google Scholar). its the technique has however from of the low reaction efficiency of the used reagents. With the of enrichable cross-linking reagents like PhoX (9Steigenberger B. Pieters R.J. Heck A.J.R. Scheltema R.A. PhoX: An IMAC-Enrichable Cross-Linking Reagent.ACS Cent. Sci. 2019; 5: 1514-1522Crossref PubMed Scopus (37) Google Scholar, A. C. Y. T. J. A. S. D. Huang L. A new in cross-linking mass spectrometry platform to interactions in Cell. Proteomics. 2014; Full Text Full Text PDF PubMed Scopus Google Scholar), this can be With these reagents the sample can be focusing only on peptides modified by the cross-linking reagent, mono-linked and cross-linked peptide are however still to the of as the mono-linked peptides not provide the structural information and typically up of the sample after Here, we the of a novel approach ion mobility to the mono-linked from the cross-linked peptides, providing to the of we present a novel technique capable of sequencing of a of mono-linked peptides, still sequencing the desired cross-linked peptides. The approach is on the timsTOF which a trapped ion mobility in a MS platform the we have that the can the between mono-linked and cross-linked peptides. This the a beneficial for complex

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