SWATH enables precise label-free quantification on proteome scale.

Abstract

MS-based proteomics has emerged as a powerful tool in biological studies. The shotgun proteomics strategy, in which proteolytic peptides are analyzed in data-dependent mode, enables a detection of the most comprehensive proteome (>10000 proteins from whole-cell lysate). The quantitative proteomics uses stable isotopes or label-free method to measure relative protein abundance. The isotope labeling strategies are more precise and accurate compared to label-free methods, but labeling procedures are complicated and expensive, and the sample number and types are also limited. Sequential window acquisition of all theoretical mass spectra (SWATH) is a recently developed technique, in which data-independent acquisition is coupled with peptide spectral library match. In principle SWATH method is able to do label-free quantification in an MRM-like manner, which has higher quantification accuracy and precision. Previous data have demonstrated that SWATH can be used to quantify less complex systems, such as spiked-in peptide mixture or protein complex. Our study first time assessed the quantification performance of SWATH method on proteome scale using a complex mouse-cell lysate sample. In total 3600 proteins got identified and quantified without sample prefractionation. The SWATH method shows outstanding quantification precision, whereas the quantification accuracy becomes less perfect when protein abundances differ greatly. However, this inaccuracy does not prevent discovering biological correlates, because the measured signal intensities had linear relationship to the sample loading amounts; thus the SWATH method can predict precisely the significance of a protein. Our results prove that SWATH can provide precise label-free quantification on proteome scale.