A Face in the Crowd: Recognizing Peptides Through Database Search
Peptide identification via tandem mass spectrometry sequence database searching is a key method in the array of tools available to the proteomics researcher. The ability to rapidly and sensitively acquire tandem mass spectrometry data and perform peptide and protein identifications has become a commonly used proteomics analysis technique because of advances in both instrumentation and software. Although many different tandem mass spectrometry database search tools are currently available from both academic and commercial sources, these algorithms share similar core elements while maintaining distinctive features. This review revisits the mechanism of sequence database searching and discusses how various parameter settings impact the underlying search. Peptide identification via tandem mass spectrometry sequence database searching is a key method in the array of tools available to the proteomics researcher. The ability to rapidly and sensitively acquire tandem mass spectrometry data and perform peptide and protein identifications has become a commonly used proteomics analysis technique because of advances in both instrumentation and software. Although many different tandem mass spectrometry database search tools are currently available from both academic and commercial sources, these algorithms share similar core elements while maintaining distinctive features. This review revisits the mechanism of sequence database searching and discusses how various parameter settings impact the underlying search. Innovations in tandem mass spectrometry (MS/MS) 1The abbreviations used are: MS/MStandem MSPTMpost-translational modification. have enabled the rapid growth of proteomics for chemists and biologists alike. In addition to the evolution of the instrument hardware to acquire spectra more rapidly and sensitively, improvements to data analysis facilitates the process of identifying and quantifying peptides and proteins for a wide user base. Researchers new to the field can benefit from a review of the fundamentals of tandem mass spectrometry sequence database searching, and even experienced proteomics researchers may profit from considerations of practical strategies to maximize information extraction from data. tandem MS post-translational modification. In a typical shotgun proteomics experiment, the sample is first denatured to enable proteolysis. An enzyme such as trypsin is applied to cleave proteins to smaller peptide components. This peptide mixture is usually then separated on a liquid chromatography (LC) column; more complex samples may necessitate prior fractionation by strong cation exchange or isoelectric focusing. Peptides are subjected to ionizing voltage as they are electrosprayed from the column, and these ions are introduced into the near-vacuum environment of the mass spectrometer. In a tandem mass spectrometry experiment, the instrument will first acquire a survey or precursor scan, also referred to as an MS scan, which measures all intact peptide ions eluting into the mass spectrometer at that given time. One or more peptide ions are selected, sequentially isolated, fragmented, and the resulting fragment ions are measured to produce an MS/MS spectrum. This process is repeated to automatically acquire MS/MS spectra on as many different peptide ions as possible throughout the LC gradient. See Fig. 1 for a schematic showing the relationship between precursor ions in the MS scans and the resulting MS/MS scans. While the MS/MS spectrum contains the peptide fragmentation pattern, the experimental peptide mass and charge state are obtained from the precursor ion measure in the MS spectrum. To clarify terminology, the intact peptide ions in the MS scans are termed precursor ions. Fragment ions are also called product ions and MS/MS spectra are referred to as product ion spectra. MS and MS/MS spectra can also referred to as MS1 and MS2 spectra, respectively. Although collision-induced dissociation (CID) is the most common way to generate product ions, others including electron capture dissociation (ECD), electron transfer dissociation (ETD), and infrared multiphoton dissociation (IRMPD) have also been developed. After data acquisition, the MS/MS spectra are searched against a protein sequence database to identify the underlying peptides and proteins represented in the acquired spectra. A 1994 paper by Mann and Wilm introduced the strategy of searching protein sequence databases using peptide sequence tags interpreted from MS/MS spectra (1Mann M. Wilm M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags.Anal. Chem. 1994; 66: 4390-4399Crossref PubMed Scopus (1318) Google Scholar). These tags of 3 to 5 consecutive amino acids were inferred by recognizing chains of amino acid mass differences between peaks. The masses flanking the tag along with the inferred sequence were then matched to peptide sequences drawn from a database of protein sequences. This approach was shown to be capable of identifying peptides with post-translational modifications (PTMs) by allowing for differences between the measured peptide mass and the calculated mass of peptides in the database. At first, tag inference was done manually so it was a relatively slow process that required some domain expertise to perform. In recent years, automated systems for inferring sequence tags have been joined by new tools to reconcile mass differences between spectra and sequences to yield new capabilities in modification and mutation identification (2Tanner S. Shu H. Frank A. Wang L.C. Zandi E. Mumby M. Pevzner P.A. Bafna V. InsPecT: identification of posttranslationally modified peptides from tandem mass spectra.Anal. Chem. 2005; 77: 4626-4639Crossref PubMed Scopus (504) Google Scholar, 3Liu C. Yan B. Song Y. Xu Y. Cai L. Peptide sequence tag-based blind identification of post-translational modifications with point process model.Bioinformatics. 2006; 22: e307-313Crossref PubMed Scopus (29) Google Scholar, 4Tabb D.L. Ma Z.Q. Martin D.B. Ham A.J. Chambers M.C. DirecTag: accurate sequence tags from peptide MS/MS through statistical scoring.J. Proteome Res. 2008; 7: 3838-3846Crossref PubMed Scopus (100) Google Scholar). Uninterpreted MS/MS database search algorithms, also introduced in 1994, rapidly became the standard method for protein identification. Eng et al. described the SEQUEST algorithm to match MS/MS spectra to peptides drawn from protein sequence databases (5Eng J.K. McCormack A.L. Yates 3rd, J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectr. 1994; 5: 976-989Crossref PubMed Scopus (5472) Google Scholar). This approach gained the ability to identify post-translational modifications the following year (6Yates 3rd, J.R. Eng J.K. McCormack A.L. Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database.Anal. Chem. 1995; 67: 1426-1436Crossref PubMed Scopus (1109) Google Scholar). With no requirement of manual interpretation of spectra prior to running searches, the ability to automate the search process increases throughput and opened the door for nonexperts to perform the analyses. Subsequent years saw the publication of both commercial and open-source tools that implemented this approach. For a detailed review of specific search tools and a collection of search engine comparisons, please see (7Kapp E. Schütz F. Overview of tandem mass spectrometry (MS/MS) database search algorithms.in: Current Protocols in Protein Science. John Wiley & Sons, Inc., 2007Google Scholar, 8Nesvizhskii A.I. Protein identification by tandem mass spectrometry and sequence database searching.Methods Mol. Biol. 2007; 367: 87-119PubMed Google Scholar, 9Brosch M. Swamy S. Hubbard T. Choudhary J. Comparison of Mascot and X!Tandem performance for low and high accuracy mass spectrometry and the development of an adjusted Mascot threshold.Mol. Cell. Proteomics. 2008; 7: 962-970Abstract Full Full PubMed Scopus Google Scholar, A. H. of MS/MS search algorithms for analysis of spectra from electron transfer dissociation Chem. PubMed Scopus Google Scholar, The of mass data and search algorithm on peptide identification in Chem. 2007; PubMed Scopus Google Scholar, Schütz F. D. Eng J.K. 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