Generation of High-Quality SWATH® Acquisition Data for Label-free Quantitative Proteomics Studies Using TripleTOF® Mass Spectrometers

Abstract

Data-independent acquisition is a powerful mass spectrometry technique that enables comprehensive MS and MS/MS analysis of all detectable species, providing an information rich data file that can be mined deeply. Here, we describe how to acquire high-quality SWATH^® Acquisition data to be used for large quantitative proteomic studies. We specifically focus on using variable sized Q1 windows for acquisition of MS/MS data for generating higher specificity quantitative data.

Keywords: Data-independent acquisitions; Mass spectrometry; Proteomics; Quantitation; SWATH acquisition; Variable windows.

Fig. 1

Build a Variable Window SWATH Method. Any window strategy can be constructed in text file format and loaded into the SWATH Acquisition method editor for automatic method building. (a) A text file describing the Q1 isolation window strategy can be constructed. (b) Load into SWATH Acquisition method editor and adjust the MS and MS/MS settings as shown. (c) Click OK to automatically build a SWATH Acquisition method

Fig. 2

Increasing Window Numbers/Decreasing Window Size Provides More Robust Peptide Detection. Using a yeast cell lysate (3 μg on column), SWATH data was collected (five replicates) in serial column mode (2 h gradient) using variable Q1 windows. The number of windows used to cover the mass range was increased from 60 to 100 windows and the reproducibility curves were plotted. The number of confident peptide detections (<1 % FDR) with ≤20 % CV further increased as the window size decreases [6]

Fig. 3

Variable Q1 Window Widths for SWATH Acquisition. To achieve better specificity in complex matrices, smaller Q1 windows are desirable especially in the m/z dense regions where many peptide precursors are measured. The m/z density histograms constructed from the TOF MS data for the proteome of interest (blue line) can be used to construct variable sized windows (red line), where the density of precursors in each of the isolation windows is equalized across the m/z range