What is Variable Window Acquisition and why does it improve results quality?
To continue to improve the quality of the SWATH® acquisition data that one can achieve in complex matrices, we want to be able to decrease the size of the Q1 window, while still maintaining full mass range coverage and optimal cycle times. One great way to do this is to use the new Variable Window Acquisition feature that is available in Analyst® TF Software 1.7. To achieve better specificity in complex matrices, smaller Q1 windows (red line) are used in the m/z dense regions where many peptide precursors are measured, wider windows are used where there are less precursors such that the entire mass range is still interrogated. The m/z density histograms (blue line) constructed from the TOF MS data for the proteome of interest (blue line) can be used to construct variable sized windows, where the density of precursors in each of the isolation windows is equalized across the m/z range. Download the Variable Window Calculator - try on your proteome.
To optimize the variable windows for your specific experiment, the first step is to determine your optimal cycle time. Typically I would go for 6-8 points across the chromatographic peak at half height to ensure good peak integration and quantitation. So determine the average peak width at height of peptides in your chromatography and divide by 6 or 7 to get your optimal cycle time. Cycle time is determined by the # of Q1 windows x the accumulation time for each step. I have tested down to accumulation times of 25 msec and see no loss in quantitative reproducibility, so next I would determine the max number of windows I can get into the computed cycle time and use that. I use 100 variable windows by 25 msec accumulation time for most of my SWATH work currently (link to my 100 SWATH variable window method below).
More information is available in the technical notes - "Improved Data Quality Using Variable Q1 Window Widths in SWATH Acquisition" "and "Evolution in SWATH Acquisition Provides Large Gainst in Quantified Proteins"