MSFragger-Labile: A Flexible Method to Improve Labile PTM Analysis in Proteomics

Abstract

•Many peptide modifications are labile, meaning they fragment during tandem MS.•Proteomics search engines often struggle to identify labile modifications.•MSFragger Labile incorporates modification fragmentation into search.•Labile searches can identify many more modified peptides than conventional searches. Posttranslational modifications of proteins play essential roles in defining and regulating the functions of the proteins they decorate, making identification of these modifications critical to understanding biology and disease. Methods for enriching and analyzing a wide variety of biological and chemical modifications of proteins have been developed using mass spectrometry–based proteomics, largely relying on traditional database search methods to identify the resulting mass spectra of modified peptides. These database search methods treat modifications as static attachments of a mass to particular position in the peptide sequence, but many modifications undergo fragmentation in tandem mass spectrometry experiments alongside, or instead of, the peptide backbone. While this fragmentation can confound traditional search methods, it also offers unique opportunities for improved searches that incorporate modification-specific fragment ions. Here, we present a new labile mode in the MSFragger search engine that provides the flexibility to tailor modification-centric searches to the fragmentation observed. We show that labile mode can dramatically improve spectrum identification rates of phosphopeptides, RNA-crosslinked peptides, and ADP-ribosylated peptides. Each of these modifications presents distinct fragmentation characteristics, showcasing the flexibility of MSFragger labile mode to improve search for a wide variety of biological and chemical modifications. Posttranslational modifications of proteins play essential roles in defining and regulating the functions of the proteins they decorate, making identification of these modifications critical to understanding biology and disease. Methods for enriching and analyzing a wide variety of biological and chemical modifications of proteins have been developed using mass spectrometry–based proteomics, largely relying on traditional database search methods to identify the resulting mass spectra of modified peptides. These database search methods treat modifications as static attachments of a mass to particular position in the peptide sequence, but many modifications undergo fragmentation in tandem mass spectrometry experiments alongside, or instead of, the peptide backbone. While this fragmentation can confound traditional search methods, it also offers unique opportunities for improved searches that incorporate modification-specific fragment ions. Here, we present a new labile mode in the MSFragger search engine that provides the flexibility to tailor modification-centric searches to the fragmentation observed. We show that labile mode can dramatically improve spectrum identification rates of phosphopeptides, RNA-crosslinked peptides, and ADP-ribosylated peptides. Each of these modifications presents distinct fragmentation characteristics, showcasing the flexibility of MSFragger labile mode to improve search for a wide variety of biological and chemical modifications. Posttranslational modifications (PTMs) of proteins play essential roles in defining and regulating protein functions (1Walsh C.T. Posttranslational Modification of Proteins: Expanding Nature’s Inventory.1st Ed. Roberts and Company Publishers, Greenwood Village, CO2006Google Scholar). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have become the preferred method for large-scale identification of proteins from biological samples, becoming known as mass spectrometry (MS)–based proteomics (2Doll S. Burlingame A.L. Mass spectrometry-based detection and assignment of protein posttranslational modifications.ACS Chem. Biol. 2015; 10: 63-71Crossref PubMed Scopus (153) Google Scholar). The ability of MS to detect and characterize posttranslational and chemical modifications of proteins at whole-proteome scales is currently unmatched and provides crucial insights into the function and regulation of proteins in biological systems. Analysis of PTMs is typically accomplished by broadly similar MS methods as those used for unmodified peptide analysis in proteomics, often with an added enrichment step to concentrate modified peptides of interest prior to LC-MS/MS analysis. Database search methods to identify the resulting tandem mass spectra are also similar to those used for unmodified peptides, as indeed, chemical artifacts and common modifications are nearly always included in such standard proteomics searches. The predominant search strategy for modified peptides assumes that modifications will remain intact on the peptide, simply shifting the mass of the amino acid to which they are connected by a fixed value (3Eng J.K. McCormack A.L. Yates J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5569) Google Scholar). Fragment ions matching the combined amino acid and modification masses are used to detect modified peptides and localize the modification within the peptide. However, many modifications experience their own fragmentation during tandem MS, causing a mismatch between the expected and observed fragment ions. Some peptides can still be identified, by matching only ions that do not contain the modification site, but many spectra of modified peptides cannot be identified, contributing to the “dark matter” of proteomics (4Chick J.M. Kolippakkam D. Nusinow D.P. Zhai B. Rad R. Huttlin E.L. et al.A mass-tolerant database search identifies a large proportion of unassigned spectra in shotgun proteomics as modified peptides.Nat. Biotechnol. 2015; 33: 743-749Crossref PubMed Scopus (281) Google Scholar, 5Kong A.T. Leprevost F.V. Avtonomov D.M. Mellacheruvu D. Nesvizhskii A.I. MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics.Nat. Met. 2017; 14: 513-520Crossref PubMed Scopus (678) Google Scholar). Labile modifications, or those that fragment instead of or in addition to peptide backbone fragmentation in tandem MS, are extremely common. The most abundant PTMs, phosphorylation (6Potel C.M. Lemeer S. Heck A.J.R. Phosphopeptide fragmentation and site localization by mass spectrometry: an update.Anal. Chem. 2019; 91: 126-141Crossref PubMed Scopus (65) Google Scholar, 7Brown R. Stuart S.S. Houel S. Ahn N.G. Old W.M. Large-scale examination of factors influencing phosphopeptide neutral loss during collision induced dissociation.J. Am. Soc. Mass Spectrom. 2015; 26: 1128-1142Crossref PubMed Scopus (10) Google Scholar, 8Everley R.A. Huttlin E.L. Erickson A.R. Beausoleil S.A. Gygi S.P. Neutral loss is a very common occurrence in phosphotyrosine-containing peptides labeled with isobaric tags.J. Proteome Res. 2017; 16: 1069-1076Crossref PubMed Scopus (18) Google Scholar) and glycosylation, are labile, along with many less abundant PTMs like sulfation (9Nemeth-Cawley J.F. Karnik S. Rouse J.C. Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry.J. Mass Spectrom. 2001; 36: 1301-1311Crossref PubMed Scopus (43) Google Scholar) and R. et fragmentation of ADP-ribosylated and their in database Am. Soc. Mass Spectrom. PubMed Scopus Google and many peptide modifications of chemical modifications used for methods et of Chem. PubMed Scopus Google and peptides a of a peptide RNA-crosslinked peptides S. PubMed Scopus Google and many many modifications of interest are labile, have been to identify and localize peptides labile modifications. methods like and typically The of in Chem. PubMed Scopus Google not always S. Burlingame A.L. fragmentation in and dissociation.J. Am. Soc. Mass Spectrom. PubMed Scopus Google modifications intact they are labile in collision methods, database search methods to identify modified peptides R. Burlingame A.L. of protein using mass spectrometry on S. PubMed Scopus Google Scholar, for peptide data analysis by of and PubMed Scopus Google Scholar). However, these methods at the of The of in Chem. PubMed Scopus Google many to for searches. for modifications that are only labile, such as the most common analysis traditional search engines neutral from a such as loss of acid from peptides J.K. an database search PubMed Scopus Google Scholar, Burlingame A.L. analysis of tandem mass spectrometry data from PubMed Scopus Google Scholar, R.A. a peptide search engine into the Proteome Res. 10: PubMed Scopus Google which for improved search of modified peptides in but the flexibility to more modifications, such as for modification that is during fragmentation Burlingame A.L. analysis of tandem mass spectrometry data from PubMed Scopus Google Scholar). the most search methods for PTMs common neutral or search methods to a particular as is common for search methods for new or less common modifications are not as the fragment ions to these modifications are not in common search search engines have been developed or to search modifications, for glycosylation, modification-specific fragment ions. These search engines and are extremely but are with a particular or of modifications, in the flexibility to new modifications or fragment ions to the an approach in the search method developed for for or peptide modifications (4Chick J.M. Kolippakkam D. Nusinow D.P. Zhai B. Rad R. Huttlin E.L. et al.A mass-tolerant database search identifies a large proportion of unassigned spectra in shotgun proteomics as modified peptides.Nat. Biotechnol. 2015; 33: 743-749Crossref PubMed Scopus (281) Google Scholar, 5Kong A.T. Leprevost F.V. Avtonomov D.M. Mellacheruvu D. Nesvizhskii A.I. MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics.Nat. Met. 2017; 14: 513-520Crossref PubMed Scopus (678) Google Scholar). and the similar mass or searches modification with Proteome Res. PubMed Scopus Google of peptides with a or a between the peptide mass and the observed mass from peptides with or known modifications to be for labile modifications, the peptide is often modifications in these methods, as a are to improve the of such searches. this the of the search for modifications, a search method in MSFragger to for fragment ions intact modification A.T. Avtonomov D.M. et of modified peptides using PubMed Scopus Google Scholar). However, the search is a for labile modifications that are from the peptide during as for peptide fragment ions the modification is the method in these The MSFragger search method of this for dramatically by for peptide backbone fragment ions glycosylation, or with a Nesvizhskii A.I. and comprehensive and analysis with Met. PubMed Scopus Google Scholar). search engines that searches incorporate or of these as such as peptide and protein identification PubMed Scopus Google Burlingame A.L. analysis of tandem mass spectrometry data from PubMed Scopus Google and modification with Proteome Res. PubMed Scopus Google Scholar). many modifications are not a of the or a of the peptide with such as the loss of from and during neutral loss of Modification loss can also or fragment ions in the mass spectrum that the of a particular modification R. Stuart S.S. Houel S. Ahn N.G. Old W.M. Large-scale examination of factors influencing phosphopeptide neutral loss during collision induced dissociation.J. Am. Soc. Mass Spectrom. 2015; 26: 1128-1142Crossref PubMed Scopus (10) Google Scholar, 8Everley R.A. Huttlin E.L. Erickson A.R. Beausoleil S.A. Gygi S.P. Neutral loss is a very common occurrence in phosphotyrosine-containing peptides labeled with isobaric tags.J. Proteome Res. 2017; 16: 1069-1076Crossref PubMed Scopus (18) Google Scholar, R. et fragmentation of ADP-ribosylated and their in database Am. Soc. Mass Spectrom. PubMed Scopus Google Scholar, Nesvizhskii A.I. for detection in spectra of modified Scopus Google Scholar, S. S. D. et of in the Met. PubMed Scopus Google Scholar, S.A. mass spectrometry of ADP-ribosylated Am. Soc. Mass Spectrom. PubMed Scopus Google Scholar, D. S.S. Neutral loss of acid in peptide a for mass identification of protein Am. Soc. Mass Spectrom. PubMed Scopus Google Scholar). a wide of labile modification we have new in to as Labile mode searches of spectra for ions a labile as as peptide or fragment masses resulting from modification fragmentation to improve spectrum identification and While are search engines that or of these for modifications, MSFragger Labile offers these for modification by making the fragment ions a of and Here, we the of labile mode searches to the spectral assignment for and We used in to the fragment ions to in labile to be for modifications fragmentation Nesvizhskii A.I. for detection in spectra of modified Scopus Google Scholar). Labile search been into MSFragger with and and for the labile phosphorylation and searches are from the in data from from the of et S. PubMed Scopus Google Scholar). by and and by LC-MS/MS on an using at collision to the Analysis data from the from the data mass from and samples, and on an mass using at et of 2019; PubMed Scopus Google Scholar). data from and to the of et from at collision and on an mass B. analysis of in 2017; Scopus Google Scholar). data from in which ADP-ribosylated peptides using from with and on an modified with a for experiments J.M. B. et using PubMed Scopus Google Scholar). data from ADP-ribosylated peptides from and using and by on an at R. S. et identification of the of and Scopus Google Scholar). to and using from D. R. D. for proteomics PubMed Scopus Google Scholar, B. R. D. S. et al.A for mass spectrometry and proteomics.Nat. Biotechnol. 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