title: SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries.
abstract: The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries.
authors.affiliation: From the ‡Australian Proteome Analysis Facility (APAF), Dept. Chemistry & Biomolecular Sciences, Macquarie University, Sydney, Australia.
authors.forename: Jemma X
authors.initials: JX
authors.lastname: Wu
authors.affiliation: From the ‡Australian Proteome Analysis Facility (APAF), Dept. Chemistry & Biomolecular Sciences, Macquarie University, Sydney, Australia.
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Song
authors.affiliation: From the ‡Australian Proteome Analysis Facility (APAF), Dept. Chemistry & Biomolecular Sciences, Macquarie University, Sydney, Australia.
authors.forename: Dana
authors.initials: D
authors.lastname: Pascovici
authors.affiliation: From the ‡Australian Proteome Analysis Facility (APAF), Dept. Chemistry & Biomolecular Sciences, Macquarie University, Sydney, Australia.
authors.forename: Thiri
authors.initials: T
authors.lastname: Zaw
authors.affiliation: From the ‡Australian Proteome Analysis Facility (APAF), Dept. Chemistry & Biomolecular Sciences, Macquarie University, Sydney, Australia.
authors.forename: Natasha
authors.initials: N
authors.lastname: Care
authors.affiliation: From the ‡Australian Proteome Analysis Facility (APAF), Dept. Chemistry & Biomolecular Sciences, Macquarie University, Sydney, Australia.
authors.forename: Christoph
authors.initials: C
authors.lastname: Krisp
authors.affiliation: From the ‡Australian Proteome Analysis Facility (APAF), Dept. Chemistry & Biomolecular Sciences, Macquarie University, Sydney, Australia mmolloy@proteome.org.au.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Molloy
mesh_terms: D002853:Chromatography, Liquid; D006801:Humans; D020014:K562 Cells; D019151:Peptide Library; D040901:Proteomics / Q000379:methods*; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D019151:Peptide Library