For research use only. Not for use in diagnostic procedures.
To optimize the variable windows for a SWATH® acquisition experiment, determine your optimal cycle time. Typically, 6-8 points across the chromatographic peak at half height are needed to ensure good peak integration and quantitation. Determine the average peak width at half height for the peptide peaks in your chromatogram and divide by 6 or 7 to get your optimal cycle time. Cycle time is determined by the number of Q1 windows multiplied by the accumulation time for each step.
Download the SWATH variable window calculator from our website here. Be sure to save a blank copy so you always have a copy of the original.
The spreadsheet scales the SWATH window size across the m/z range depending on the number of precursors found in that m/z range. That is, the Q1 window widths are varied over the m/z range so that each window contains a constant density from an intensity weighted histogram of the survey scan data versus m/z.
To use the variable window calculator, follow the directions below:
1. Fill in the values as shown:
2. In PeakView® software, open an IDA method that was collected on your target proteome with a gradient length similar to what was used in your SWATH study. Ensure that this file has a TOF MS scan that encompasses the mass range that will be built in the SWATH variable window experiment.
3. Open the TOF MS TIC (blue
) and then select the time range in which the peptides elute. Double click on the selection (red box
) to extract a single TOF MS spectrum of all precursors within that time range (third panel
4. In the SWATH calculator spreadsheet, open the Variable Window Calculator and paste the data list above in the INPUT – PeakView ave spectrum tab for the worksheet. The m/z data should be in column A and the intensity data in column B. The length of the data series does not matter as long as it does not exceed Excel's row limit of approximately one million rows.
5.Start the calculations on the data in the whole workbook by hitting F9. Be sure to wait for the calculations to finish completely. This could take several minutes.
6. Paste the three columns from the OUTPUT for Analyst tab into a new Excel workbook and save as a tab-delimited text file. This text file can be imported directly into the SWATH Acquisition method editor in Analyst® Software.
7. Run the Analyst method for SWATH variable windows. The maximum number of windows to use for this experiment depends on the type of TripleTOF® system in use:
- For the TripleTOF 5600+ System: 100 windows
- For the TripleTOF 6600 System: 200 windows
A graph of the m/z histogram and the variable window strategy calculated are shown on the Visualization of Var Win Assay tab.