SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries
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SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries. / Wu, Jemma X; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P.
In: MOL CELL PROTEOMICS, Vol. 15, No. 7, 07.2016, p. 2501-14.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries
AU - Wu, Jemma X
AU - Song, Xiaomin
AU - Pascovici, Dana
AU - Zaw, Thiri
AU - Care, Natasha
AU - Krisp, Christoph
AU - Molloy, Mark P
N1 - © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2016/7
Y1 - 2016/7
N2 - 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.
AB - 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.
KW - Chromatography, Liquid
KW - Humans
KW - K562 Cells
KW - Peptide Library
KW - Proteomics
KW - Software
KW - Tandem Mass Spectrometry
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.1074/mcp.M115.055558
DO - 10.1074/mcp.M115.055558
M3 - SCORING: Journal article
C2 - 27161445
VL - 15
SP - 2501
EP - 2514
JO - MOL CELL PROTEOMICS
JF - MOL CELL PROTEOMICS
SN - 1535-9476
IS - 7
ER -