Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis

Abstract

Most proteomic studies use liquid chromatography coupled to tandem mass spectrometry to identify and quantify the peptides generated by the proteolysis of a biological sample. However, with the current methods it remains challenging to rapidly, consistently, reproducibly, accurately, and sensitively detect and quantify large fractions of proteomes across multiple samples. Here we present a new strategy that systematically queries sample sets for the presence and quantity of essentially any protein of interest. It consists of using the information available in fragment ion spectral libraries to mine the complete fragment ion maps generated using a data-independent acquisition method. For this study, the data were acquired on a fast, high resolution quadrupole-quadrupole time-offlight (TOF) instrument by repeatedly cycling through 32 consecutive 25-Da precursor isolation windows (swaths). This SWATH MS acquisition setup generates, in a single sample injection, time-resolved fragment ion spectra for all the analytes detectable within the 400 -1200 m/z precursor range and the user-defined retention time window. We show that suitable combinations of fragment ions extracted from these data sets are sufficiently specific to confidently identify query peptides over a dynamic range of 4 orders of magnitude, even if the precursors of the queried peptides are not detectable in the survey scans. We also show that queried peptides are quantified with a consistency and accuracy comparable with that of selected reaction monitoring, the gold standard proteomic quantification method. Moreover, targeted data extraction enables ad libitum quantification refinement and dynamic extension of protein probing by iterative re-mining of the once-and-forever acquired data sets. This combination of unbiased, broad range precursor ion fragmentation and targeted data extraction alleviates most constraints of present proteomic methods and should be equally applicable to the comprehensive analysis of other classes of analytes, beyond proteomics. Molecular & Cellular Proteomics 11: 10.1074/mcp.O111.016717, 1-17, 2012. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)1 is considered the method of choice for the identification and quantification of proteins and proteomes (1-4) and for the analysis of metabolites, lipids, glycans, and many other types of (bio)molecules. For proteomics, two main LC-MS/MS strategies have been used thus far. They have in common that the sample proteins are converted by proteolysis into peptides, which are then separated by (capillary) liquid chromatography. They differ in the mass spectrometric method used. The first and most widely used strategy is known as shotgun or discovery proteomics. For this method, the MS instrument is operated in data-dependent acquisition (DDA) mode, where fragment ion (MS2) spectra for selected precursor ions detectable in a survey (MS1) scan are generated (5). The resulting fragment ion spectra are then assigned to their corresponding peptide sequences by sequence data...

Systems pathology analysis identifies neurodegenerative nature of age‐related vitreoretinal interface diseases

“…Sequential window acquisition of all theoretical mass spectra (SWATH MS) is a recently developed, massively parallel protein targeting technique that features high accuracy and high reproducibility in protein quantification (Gillet et al, 2012). To validate our findings, we performed SWATH MS analysis of vitreous samples from 5 iERM, 5 MH and 4 DME patients.…”

Characterization of the porcine seminal plasma proteome comparing ejaculate portions

“…For SWATH-MS-based experiments the instrument (SCIEX TripleTOF 5600) was configured as described by Gillet et al [17]. Briefly, the mass spectrometer was operated in a looped product ion mode.…”

“…To achieve this, SP-samples from boars with proven fertility would be examined using 1-D SDS PAGE and Nano LC-ESI-MS/MS followed by bioinformatics of Gene Ontology (GO) to evaluate cellular location, molecular function and biological process of the identified proteins. In addition, the boar SPproteome will be described so that a spectral library can be built for relative 'label free' protein quantitation with SWATH approach [17].…”

Applications of targeted proteomics in systems biology and translational medicine

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