Finding Chimeras: a Bioinformatics Strategy for Identification of Cross-linked Peptides

Chemical cross-linking, followed by identification of the cross-linked residues, is a powerful approach to probe the topologies and interacting surfaces of protein assemblies. In this work, we demonstrate a new bioinformatics approach using multiple program modules within the software package “Protein Prospector” that greatly facilitates the discovery of cross-linked peptides in chemical cross-linking studies. Examples are given for how this approach has been used for defining interfaces in heterodimeric and homodimeric protein complexes, both of which provide results in close agreement with crystal structures, verifying the reliability of the approach. Chemical cross-linking, followed by identification of the cross-linked residues, is a powerful approach to probe the topologies and interacting surfaces of protein assemblies. In this work, we demonstrate a new bioinformatics approach using multiple program modules within the software package “Protein Prospector” that greatly facilitates the discovery of cross-linked peptides in chemical cross-linking studies. Examples are given for how this approach has been used for defining interfaces in heterodimeric and homodimeric protein complexes, both of which provide results in close agreement with crystal structures, verifying the reliability of the approach. Proteins act in the context of dynamic protein complexes and interaction networks (1Gavin A.C. Aloy P. Grandi P. Krause R. Boesche M. Marzioch M. Rau C. Jensen L.J. Bastuck S. Dümpelfeld B. Edelmann A. Heurtier M.A. Hoffman V. Hoefert C. Klein K. Hudak M. Michon A.M. Schelder M. Schirle M. Remor M. Rudi T. Hooper S. Bauer A. Bouwmeester T. Casari G. Drewes G. Neubauer G. Rick J.M. Kuster B. Bork P. Russell R.B. Superti-Furga G. 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To gain mechanistic insights into various cellular processes, it is important to devise enabling strategies for the mapping of protein interaction surfaces. Chemical cross-linking has been an established method to study protein interaction partners for decades (3Wong S.S. Chemistry of Protein Conjugation and Cross-linking. CRC Press, Boca Raton, FL1991Google Scholar) and has been recently revived by conjunctional mass spectrometric analysis of the cross-linking reaction products (4Sinz A. Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions.Mass Spectrom. Rev. 2006; 25: 663-682Crossref PubMed Scopus (525) Google Scholar). Mass spectrometric identification of the cross-linked residues provides valuable spatial restraints in defining protein interacting surfaces. Nevertheless, comprehensive analysis of proteolytic digests of cross-linked protein assemblies is challenging. The difficulty is largely due to the fact that most generic cross-linking reagents react with multiple residues and meanwhile undergo competing side reactions (with solvent, for example), inactivating one of the reactive groups. This means that the dominant reaction products are where only one end of the reagent reacts with a peptide, and these are referred to as dead-end modified peptides (5Schilling B. Row R.H. Gibson B.W. Guo X. Young M.M. MS2Assign, automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides.J. Am. Soc. Mass Spectrom. 2003; 14: 834-850Crossref PubMed Scopus (234) Google Scholar). To facilitate the detection and separation of cross-linked species, various moieties have been introduced into the cross-linking reagents including fluorophores (6Sinz A. Wang K. Mapping protein interfaces with a fluorogenic cross-linker and mass spectrometry: application to nebulin-calmodulin complexes.Biochemistry. 2001; 40: 7903-7913Crossref PubMed Scopus (64) Google Scholar), isotope tags (7Müller D.R. Schindler P. Towbin H. Wirth U. Voshol H. Hoving S. Steinmetz M.O. Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis.Anal. Chem. 2001; 73: 1927-1934Crossref PubMed Scopus (187) Google Scholar, 8Pearson K.M. Pannell L.K. Fales H.M. Intramolecular cross-linking experiments on cytochrome c and ribonuclease A using an isotope multiplet method.Rapid Commun. Mass Spectrom. 2002; 16: 149-159Crossref PubMed Scopus (104) Google Scholar), cleavable sites (9Bennett K.L. Kussmann M. Björk P. Godzwon M. Mikkelsen M. Sørensen P. Roepstorff P. Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping—a novel approach to assess intermolecular protein contacts.Protein Sci. 2000; 9: 1503-1518Crossref PubMed Scopus (133) Google Scholar), affinity handles (10Alley S.C. Ishmael F.T. Jones D. Benkovic S.J. Mapping protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme using a novel trifunctional photo-cross-linking and affinity reagent.J. Am. Chem. Soc. 2000; 122: 6126-6127Crossref Scopus (66) Google Scholar, 11Hurst G.B. Lankford T.K. Kennel S.J. Mass spectrometric detection of affinity purified crosslinked peptides.J. Am. Soc. Mass Spectrom. 2004; 15: 832-839Crossref PubMed Scopus (62) Google Scholar, 12Trester-Zedlitz M. Kamada K. Burley S.K. Fenyö D. Chait B.T. Muir T.W. A modular cross-linking approach for exploring protein interactions.J. Am. Chem. Soc. 2003; 125: 2416-2425Crossref PubMed Scopus (161) Google Scholar, 13Itoh Y. Cai K. Khorana H.G. Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: use of a chemically preactivated reagent.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 4883-4887Crossref PubMed Scopus (102) Google Scholar, 14Cai K. Itoh Y. Khorana H.G. Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 4877-4882Crossref PubMed Scopus (137) Google Scholar), and a benzyl marker for tandem mass spectrometric analysis (15Back J.W. Hartog A.F. Dekker H.L. Muijsers A.O. de Koning L.J. de Jong L. A new crosslinker for mass spectrometric analysis of the quaternary structure of protein complexes.J. Am. Soc. Mass Spectrom. 2001; 12: 222-227Crossref PubMed Scopus (78) Google Scholar). However, mere incorporation of these moieties cannot distinguish the cross-linked peptides fromdead-end modified products (16Chu F. Mahrus S. Craik C.S. Burlingame A.L. Isotope-coded and affinity-tagged cross-linking (ICATXL): an efficient strategy to probe protein interaction surfaces.J. Am. Chem. Soc. 2006; 128: 10362-10363Crossref PubMed Scopus (50) Google Scholar). The high complexity of these samples requires tandem mass spectrometric fragmentation data for confident identification of the cross-linked peptides. Although highly desirable, available bioinformatics tools for automatic analysis of fragmentation spectra of the cross-linked samples are far from robust, mainly limited by the difficulty in identifying fragments that contain components from cross-linked peptide species. Thus far, most of the reported software either rely on prescreening for cross-linker-containing peptides by their distinct isotope pattern (17Rinner O. Seebacher J. Walzthoeni T. Mueller L.N. Beck M. Schmidt A. Mueller M. Aebersold R. Identification of cross-linked peptides from large sequence databases.Nat. Methods. 2008; 5: 315-318Crossref PubMed Scopus (5) Google Scholar, 18Gao Q. Xue S. Doneanu C.E. Shaffer S.A. Goodlett D.R. Nelson S.D. Pro-CrossLink. Software tool for protein cross-linking and mass spectrometry.Anal. Chem. 2006; 78: 2145-2149Crossref PubMed Scopus (54) Google Scholar, 19Maiolica A. Cittaro D. Borsotti D. Sennels L. Ciferri C. Tarricone C. Musacchio A. Rappsilber J. Structural analysis of multiprotein complexes by cross-linking, mass spectrometry, and database searching.Mol. Cell. Proteomics. 2007; 6: 2200-2211Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) or require manual entry of peak lists for each predetermined cross-linked peptide candidate (5Schilling B. Row R.H. Gibson B.W. Guo X. Young M.M. MS2Assign, automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides.J. Am. Soc. Mass Spectrom. 2003; 14: 834-850Crossref PubMed Scopus (234) Google Scholar). Another strategy has just been reported where mass modification searching allowed identification of cross-linked candidates (20Singh P. Shaffer S.A. Scherl A. Holman C. Pfuetzner R.A. Larson Freeman T.J. Miller S.I. Hernandez P. Appel R.D. Goodlett D.R. Characterization of protein cross-links via mass spectrometry and an open-modification search strategy.Anal. Chem. 2008; 80: 8799-8806Crossref PubMed Scopus (63) Google Scholar). It is based on selecting quadruply charged precursors as potentially being cross-linked peptides and then searching the obtained spectra one at a time to try to determine whether they are cross-linked peptides and identify the sequences involved. Here we present an alternative approach using available programs in the bioinformatics software package “Protein Prospector” (21Chalkley R.J. Baker P.R. Huang L. Hansen K.C. Allen N.P. Rexach M. Burlingame A.L. Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer: II. new developments in Protein Prospector allow for reliable and comprehensive automatic analysis of large datasets.Mol. Cell. Proteomics. 2005; 4: 1194-1204Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 22Chalkley R.J. Baker P.R. Medzihradszky K.F. Lynn A.J. Burlingame A.L. In-depth analysis of tandem mass spectrometry data from disparate instrument types.Mol. Cell. Proteomics. 2008; 7: 2386-2398Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), which allows analysis of all MS/MS spectra acquired in a data set in one analysis. Thus, one can identify unmodified and cross-linked peptides together in a single search result. In all cross-linked protein complexes tested, our new strategy has led to the discovery of not only all cross-linked peptides previously identified through manual analysis but new cross-linked peptides as well. These comprehensive results with the and comprehensive of the approach to protein assemblies. to the study of the complex are in a F. F. P. Burlingame A.L. the of and by using chemical cross-linking and tandem mass Natl. Acad. Sci. U.S.A. 2004; PubMed Scopus Google Scholar). used from modified from The used for the of as previously M.D. K.C. Mahrus S. Craik C.S. Burlingame A.L. identified in the Sci. 2001; PubMed Scopus Google Scholar). and as previously F. G. Burlingame A.L. Identification of novel quaternary interactions in the Sci. 2006; 15: PubMed Scopus Google Scholar). separation of the cross-linked both and and and the to differential a of the into an via a and by a at a of The to the of a mass data acquired using the software lists using the and then using within Protein the database with to which the sequences of and peptides with to and mass of and and the only protein and peptide to Protein Prospector as previously (21Chalkley R.J. Baker P.R. Huang L. Hansen K.C. Allen N.P. Rexach M. Burlingame A.L. Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer: II. new developments in Protein Prospector allow for reliable and comprehensive automatic analysis of large datasets.Mol. Cell. Proteomics. 2005; 4: 1194-1204Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) where a given is for each with the on the a an These then to as previously R.J. Baker P.R. Medzihradszky K.F. Lynn A.J. Burlingame A.L. In-depth analysis of tandem mass spectrometry data from disparate instrument types.Mol. Cell. Proteomics. 2008; 7: 2386-2398Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) by the of for and a and then of a given being in this The of for all identified in the then used for a In this modification to and the protein of in the automated peak a mass of that is not on to allow of spectra where the isotope as the as these this search allowed for a single mass modification of between and to residues or the peptide mass to used to the mass of a of of the of an search the as for the a reported of results then that to peptides with a large mass modification then using program in Protein The to of the of cross-linked peptides the sequences of the cross-linked used in in cross-linking of the and to The program a of or cross-linked peptide that to the identified one of the cross-linked peptides from the this for the cross-linked peptide, and the sequence can into the of the results to peak to the peptides in the for an is with the of this peptide, the an whether they the peptide assignment based on the of for the of the peptide by and how from the assignment of the peptide are as fragments from the we an mass modification searching in a program in the Protein Prospector to identify peptide R.J. Baker P.R. Medzihradszky K.F. Lynn A.J. Burlingame A.L. In-depth analysis of tandem mass spectrometry data from disparate instrument types.Mol. Cell. Proteomics. 2008; 7: 2386-2398Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). that it a powerful tool for identifying cross-linked peptides by the cross-linked peptide as a single peptide with a large modification of mass R.J. Baker P.R. Medzihradszky K.F. Burlingame A.L. of the on Mass and for Mass Scholar) is in proteolytic peptides of a cross-linked protein complex are by of the protein complex are identified on the of unmodified peptides by protein database searching This of is then used as a database for an mass modification for peptides with large which are potentially cross-linked peptide can a database to a of reliability for for by searching a database of a database and a of the database database can using Protein Prospector J.E. search strategy for in protein by mass Methods. 2007; 4: PubMed Scopus Google identification of the peptide peptide in a cross-linked the Protein Prospector program F. F. P. Burlingame A.L. the of and by using chemical cross-linking and tandem mass Natl. Acad. Sci. U.S.A. 2004; PubMed Scopus Google Scholar, F. G. Burlingame A.L. Identification of novel quaternary interactions in the Sci. 2006; 15: PubMed Scopus Google Scholar) is to all cross-linked peptide from the in the database that the mass The cross-linked peptide peptide is based on the sequence of the peptide peptide identified from the The fragmentation of the cross-linked is then in fragmentation of both peptide components using which allows to multiple sequences for an This allows of identification of the cross-linked To assess to identify as components present as and high a as of the reported as are of this new we it to a previously the heterodimeric complex of The interaction between and protein of and to the P. to structure of the 2005; 15: PubMed Scopus Google Scholar). previously a chemical cross-linking study of the complex and identified cross-linked peptides that only present in the digests of the cross-linked complex F. F. P. Burlingame A.L. the of and by using chemical cross-linking and tandem mass Natl. Acad. Sci. U.S.A. 2004; PubMed Scopus Google Scholar). the data set using the new bioinformatics approach. In an mass modification we identified peptides with large mass to a peptide by a to the of the database The of the cross-linked peptide components then using these cross-linked species, of are interacting surfaces of the the are cross-links from either or In a of data all cross-linked peptides in the T. data set that previously identified through manual using the new Protein Prospector In new cross-links reported and by manual The discovery of cross-links is not data analysis on peptides that only in the cross-linked However, it the of our which has for protein assemblies where cross-linking for protein components and to protein assemblies are Chemical cross-linking on complexes an and cross-links are from their In a we a differential which but not cross-linked as to allow of and cross-links F. G. Burlingame A.L. Identification of novel quaternary interactions in the Sci. 2006; 15: PubMed Scopus Google Scholar). the program in Protein Prospector allows of peptides identified in it can used for of protein assemblies. To demonstrate we a cross-linking study of a homodimeric M.D. K.C. Mahrus S. Craik C.S. Burlingame A.L. identified in the Sci. 2001; PubMed Scopus Google Scholar), using an with an and to used for this from by and both samples through the including and followed by mass modification searching and analysis. cross-linked mass modification searching in it is to distinguish from cross-links at this we the identified peptides between of the and samples in which led to the identification of cross-links an the mass modification search reported a as the peptide with a mass of on then a of all cross-linked peptide from the protein that to the mass of this cross-linked species. that one the peptide identified in the mass modification allowed the peptide protein has an of the is residues the of the protein sequence in the to identified as the peptide This identification by from the peptide the we a in mass the cross-linked and identified fragmentation from both peptide the cross-linker and are and in the cross-linked Thus, the mass is due to the cross-linker of of has led to the formation of this cross-linked species. Although this not provide on the it the of this mass modification searching that of cross-linked products from this However, identified in the database is that these components for MS/MS analysis. The cross-links are the structure of in The cross-linking results are in agreement with both the crystal structure and a study on this M.D. K.C. Mahrus S. Craik C.S. Burlingame A.L. identified in the Sci. 2001; PubMed Scopus Google Scholar). Chemical cross-linking to protein interaction surfaces are most on complexes as in this However, the approach to the analysis of complexes and To an of how this bioinformatics approach in a complex the peak lists from the complex data set combined with a peak from a available protein data set J. J.S. L. J. A. S. P.R. J.E. P. H. Schmidt A. R. Aebersold R. The protein a data set to in the of and protein identification software Proteome 2008; 7: PubMed Scopus Google Scholar), acquired on a mass This just the analysis of the combined data set provides of the of this bioinformatics approach to identify the cross-linked in the of and peptides. search of this data set mass a of identified including and mass modification searching in then on the combined data set a database of these and of of the results that the spectra identified as cross-linked in analysis only and all identified to the peptide and mass However, the in the of Thus, reported to results However, the is at high with a set at a of spectra with mass reported to either or and all of these to spectra in the cross-linking data spectra from the protein the fact that as spectra in the data set as the cross-linking data for of these to or of the peptide but the peak to the search in the of the and peptide given the these spectra both to unmodified of peptides the and the at a that an of the peptide as reported in the mass modification search but and residues introduced into the to try to These results that the software is at from results but that the for the have This is by the of into that is by the of a of the mass a given by the of peptides in the database that have the modification a large mass is then the of peptides is large in with the of the identifying the cross-linked at searching for mass only the cross-linked protein sequences peptide in the database precursors which is a the of unmodified peptides of the mass in the However, this in of precursors is the for all spectra whether or it a in for all results whether or the use of a database searching strategy and then of a based on these results an identified cross-linked peptide the in the is the use of to try to the peptide present in the cross-linked The of only a on the to identify the for the cross-linked peptides for of reported that only one peptide of the that from the in the for peptide and for one to we have a bioinformatics approach for the analysis of cross-linked protein The mass modification searching is comprehensive and and all of peptides including unmodified dead-end modified and and cross-linked In the that contain cross-linked side are identifying the cross-linked peptide our approach cross-linked peptides with and that only unmodified However, it not that contain the cross-linker but fragments of both peptide (5Schilling B. Row R.H. Gibson B.W. Guo X. Young M.M. MS2Assign, automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides.J. Am. Soc. Mass Spectrom. 2003; 14: 834-850Crossref PubMed Scopus (234) Google Scholar). of these a of the This strategy has allowed the identification of a large of cross-linked peptides in all protein assemblies we have This of in data analysis is important the identification of multiple cross-links within a complex valuable for of protein interaction surfaces. mass modification searching is for including the study of on and complexity introduced by these The of purified protein complexes, but we a complex and that the bioinformatics strategy cross-linked products in a complex The this approach with complexity is the fact that the of cross-linked with of our strategy of the complex at a the in at each is only a The where the in protein candidates has the most are one of the peptides in the cross-linked complex is of this can in it is to determine the sequence of the cross-linked peptide, it not to it to a the mass of of the cross-linked peptides with a of cross-linking reported in high and mass data for large protein The strategy not the to the peptide, which highly for a of However, with our mass modification strategy it to a search where identified one of the peptides we a modification of the mass to the peptide, which then allow the peptide to identified in a database searching This is a we are to in a of the A of all cross-linking is that the products of are highly The results most bioinformatics on the precursors of being for In our of a complex data we the of peptide but we not the that are unmodified peptides in the then the of the cross-linked peptides of being for fragmentation analysis are It is to the of precursors for fragmentation cross-linked products by only precursors of cross-linked peptide products have and a in each peptide, they most quadruply unmodified peptides have this high However, an strategy for modified peptides by a into the cross-linker the most approach (10Alley S.C. Ishmael F.T. Jones D. Benkovic S.J. Mapping protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme using a novel trifunctional photo-cross-linking and affinity reagent.J. Am. Chem. Soc. 2000; 122: 6126-6127Crossref Scopus (66) Google Scholar, 11Hurst G.B. Lankford T.K. Kennel S.J. Mass spectrometric detection of affinity purified crosslinked peptides.J. Am. Soc. Mass Spectrom. 2004; 15: 832-839Crossref PubMed Scopus (62) Google Scholar, 12Trester-Zedlitz M. Kamada K. Burley S.K. Fenyö D. Chait B.T. Muir T.W. A modular cross-linking approach for exploring protein interactions.J. Am. Chem. Soc. 2003; 125: 2416-2425Crossref PubMed Scopus (161) Google Scholar, 13Itoh Y. Cai K. Khorana H.G. Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: use of a chemically preactivated reagent.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 4883-4887Crossref PubMed Scopus (102) Google Scholar, 14Cai K. Itoh Y. Khorana H.G. Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 4877-4882Crossref PubMed Scopus (137) Google Scholar). In our new bioinformatics approach and software tools a in the to from chemical cross-linking and use of this of strategy for protein complex The Protein Prospector software is available on the at Mahrus and S. Craik at the of for for this with

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