Project PXD002836



SWATH mass spectrometry performance using extended ion assay libraries


The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed using peptide MS/MS reference ion 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 shared 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 using a local seed library and delivers statistical analysis of SWATH-based sample comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at different ratios to simulate common protein abundance change comparisons. SWATH-MS data with 2, 5 and 10% of yeast peptides spiked into the human cell lysate were assessed using several local and repository-based assay libraries of different complexities and proteome compositions. We evaluated detection specificity and accuracy to detect differentially abundant proteins and reporting thresholds for statistical analyses. We demonstrate that extended assay libraries integrated with local seed libraries achieve better performance than local limited assay libraries alone from the aspects of the number of peptides and proteins identified and the specificity to detect differentially abundant proteins; the performance of extended assay libraries heavily depend on the similarity of the seed and add-on libraries; statistical analysis with multiple testing correction can improve the statistical rigor needed when using large, extended assay libraries.

Sample Processing Protocol

Tryptic peptides of whole-cell protein extracts from yeast (Saccharomyces cerevisiae) and human (K562) cells were purchased from Promega (Cat # V7461 and V6951). Both extracts had been reduced with Dithiothreitol and alkylated with Iodoacetamide and digested with Trypsin/Lys-C mix. The yeast and human samples were reconstituted in 0.1% formic acid at 0.1µg/µL concentrations and stored in 10µL aliquots. To make yeast-spiked-human samples, appropriate amount of yeast protein digest were added into 1µg human protein digest, making three groups of samples that contained 2%, 5% and 10% yeast (Wyeast/Whuman x 100%) respectively in 0.05 µg/µL of human protein digest.

Data Processing Protocol

IDA MS/MS data were subjected to database searches by ProteinPilot (V5.0, AB Sciex) using the Paragon algorithm. Human (Homo sapiens) and yeast (Saccharomyces cerevisiae) reviewed protein databases were downloaded from UniProtKB (August 2014 version) and the two downloaded databases were merged making a new Yeast-Human database with 43389 entries. This Yeast-Human database was used for database searches. The search parameters were as follows: sample type: identification; cys alkylation: iodoacetamide; digestion: trypsin; special factors: none; ID focus: allow biological modifications. The group files from the database searches were loaded to PeakView (V2.1 with SWATH Quantitation plug-in) and exported as libraries in CSV format. PeakView V2.1 with SWATH quantitation plug-in (SCIEX) was used to extract SWATH MS peak areas with each of the libraries in our study. Shared and modified peptides were excluded. Other peakview SWTAH peak extraction parameters are: 100 peptides for each protein, 6 ions for each peptide, peptide FDR <1%, mass tolerance 75 ppm, retention time window 10 min.


Xiaomin Song, APAF
Xiaomin Song, Australian Proteome Analysis Facility, Macquarie University ( lab head )

Submission Date


Publication Date



TripleTOF 5600


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Experiment Type



    Wu JX, Song X, Pascovici D, Zaw T, Care N, Krisp C, Molloy MP. SWATH mass spectrometry performance using extended peptide MS/MS assay libraries. Mol Cell Proteomics. 2016 May 9. pii: mcp.M115.055558 PubMed: 27161445