title: Whole cell, label free protein quantitation with data independent acquisition: quantitation at the MS2 level.
abstract: Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is a technique that has seen limited use due to the stochastic nature of data dependent acquisition (DDA). However, data independent acquisition has the potential to make large scale MS2 quantitation a more viable technique. In this study we used an implementation of data independent acquisition--SWATH--to perform label free protein quantitation in a model bacterium Clostridium stercorarium. Four tryptic digests analyzed by SWATH were probed by an ion library containing information on peptide mass and retention time obtained from DDA experiments. Application of this ion library to SWATH data quantified 1030 proteins with at least two peptides quantified (∼ 40% of predicted proteins in the C. stercorarium genome) in each replicate. Quantitative results obtained were very consistent between biological replicates (R(2) ∼ 0.960). Protein quantitation by summation of peptide fragment signal intensities was also highly consistent between biological replicates (R(2) ∼ 0.930), indicating that this approach may have increased viability compared to recent applications in label free protein quantitation. SWATH based quantitation was able to consistently detect differences in relative protein quantity and it provided coverage for a number of proteins that were missed in some samples by DDA analysis.
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, Winnipeg, Manitoba, Canada.
authors.forename: Peter
authors.initials: P
authors.lastname: McQueen
authors.forename: Vic
authors.initials: V
authors.lastname: Spicer
authors.forename: John
authors.initials: J
authors.lastname: Schellenberg
authors.forename: Oleg
authors.initials: O
authors.lastname: Krokhin
authors.forename: Richard
authors.initials: R
authors.lastname: Sparling
authors.forename: David
authors.initials: D
authors.lastname: Levin
authors.forename: John A
authors.initials: JA
authors.lastname: Wilkins
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods; D003013:Clostridium / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments
keywords: Bottom-up proteomics; Data dependent acquisition; Data independent acquisition; Label free quantitation; Relative protein quantitation; SWATH; Technology