Robust optimization of SWATH-MS workflow for human blood serum proteome analysis using a quality by design approach

Abstract Background It is not enough to optimize proteomics assays. critical those assays are robust operating conditions. Without assays, proteomic biomarkers unlikely translate readily into the clinic. This study outlines a structured approach identification of window for and applies that method Sequential Window Acquisition all Theoretical Spectra Mass Spectroscopy (SWATH-MS). Methods We used sequential quality by design exploiting fractional screening first identify SWATH-MS parameters, then using response surface methods with good reproducibility, before validating settings in separate validation study. Results The experiment identified two parameters. modelled number proteins reproducibility as function parameters identifying an permitting maximization quantified human serum. In study, these were shown give proteome-wide coverage high quantification reproducibility. Conclusions Using experiments permits SWATH-MS. gives understanding greater data-driven confidence performance.