Variable window acquisition for SWATH analysis has enabled comprehensive analysis of complex matrices while maintaining the acquired mass range without affecting cycle times. Variable SWATH windows allow smaller Q1 windows in m/z dense regions where many peptide precursors are measured, and wider windows where fewer precursors are measured. This results in superior quantitative data and increased peptide identifications. The original SWATH method used 32 fixed windows, with 64 fixed windows now relatively common. More recently, 100 variable windows have been advocated. However, increasing the number of windows comes at a cost to either cycle time or points across a chromatographic peak, both of which have an impact on the quality of data acquired.
To maintain the number of points across a peak, thereby maintaining quantitative reproducibility when the total number of SWATH windows are increased, cycle time would need to be decreased. However, decreasing the cycle time negatively affects the quality of spectra acquired which could lead to a reduction in peptides identified and quantified.
Alternatively, keeping cycle time constant while increasing the number of SWATH windows results in fewer points across a peak that may negatively affect quantitation reproducibility by increasing variability.
For this study, a number of variables were tested including; cycle time, collision energy spread, and points across a peak. The aim was to achieve the maximum numbers of peptides and proteins identified reproducibly across a 90-min LC run, whilst finding the optimal balance between cycle time and points across a peak.