A Face in the Crowd: Recognizing Peptides Through Database Search

Abstract

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. Molecular & Cellular Proteomics 10: 10.1074/ mcp.R111.009522, 1-9, 2011.Innovations in tandem mass spectrometry (MS/MS) 1 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.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 ...

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