Recent advances of MS technologies promote an increase in the number of proteins or peptides identified and quantified. For biomarker discovery the sequential window acquisition of all theoretical fragment ion spectra (SWATH) is a strong tool for label-free proteome quantification with data-independent acquisition (DIA). In the SWATH, a reference library created by data-dependent acquisition (DDA) runs is used for identification of proteins and peptides measured in the DIA by correlating the quantitative DIA data obtained from the fragment ion spectra data with the library information for peptide identification. Although the quality and coverage of the reference library are essential for SWATH data processing and the quality of data independent acquisition (DIA) is also important, there are several problems to be solved for the ideal accurate label-free proteome quantification by SWATH.
We analyzed proteins extracted from human urine and performed SWATH DDA and DIA analysis by LC-MS (TripleTOF 6600, SCIEX). The libraries were made by using different numbers of DDA data at different conditions. The SWATH analysis data obtained with these different libraries were compared each other to point out some problems for accurate quantification.
In this presentation, we will point out the several problems of the SWATH method and propose suggestions for improvement of the SWATH data accuracy. Our study may give an insight into how to make a better library and perform SWATH analysis more accurately.