MS/MS^ALL with SWATH acquisition is a data-independent acquisition strategy that generates high resolution MS/MS spectra for all detectable analytes in a biological sample within a pre-defined mass range. This technique enables high specificity MRM-like quantitation by extracting high resolution product ions of target analytes.

To evaluate SWATH as a proteome profile differential analysis technique, we acquired two sets of SWATH-MS data using two TripleTOF 5600 mass spectrometers for samples of human K562 whole cell protein digest spiked with 0.1%, 0.5%, 2%, 5% and 10% yeast protein digest. We extracted SWATH data using single IDA run library, comprehensive sample and instrument specific library, comprehensive sample specific library from other instrument, as well as publicly available generic libraries. We tested the effect of using different libraries to the quantitation results and determined the cut-off thresholds which would generate most true positives and most true negatives with least false positives reports for differential expressed protein discovery. The quantitation accuracy was also examined.

Our result demonstrated that it is feasible to extract SWATH data using comprehensive SWATH library built from IDA data acquired on different instruments or from publicly available MS/MS data. For our tested human protein spiked with 2% and 10% yeast samples, when a comprehensive generic library was used to extract SWATH-MS data and a cut-off threshold of protein fold change larger than 1.5 with protein area T-Test P-value smaller than 0.05, 3785 human proteins were quantified correctly as not changing in quantities between samples and 155 yeast proteins were correctly quantified as being changed in quantities with false positive discovery rate of 1.37%. We observed that false positive discovery rate would be significantly reduced with more stringent cut-off criteria that measure peptide area T-Test P-values.