How Are SWATH™ Acquisition Files Recalibrated?


日付: 08/24/2017
カテゴリー: Academia Omics , Analyst Software , SWATH Acquisition

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For research use only. Not for use in diagnostic procedures.


Answer

To recalibrate a SWATH™ acquisition file:

Open a representative sample in PeakView® software, click Show > Total Ion Chromatogram (TIC), and then determine the SWATH acquisition window that the precursor m/z of your chosen cal peptide falls in. Make a note of the experiment number.User-added image
  • Select Process > Recalibrate Samples.
  • Click Open Samples…
  • Open a single sample to test the recalibration.
  • In the Experiment field, select the previously noted experiment number.
  • Set the XIC Mass Width to 0.05 Da.
  • Set the Spectral Peak Tolerance. This setting will depend on your worst calibrated sample. If the tolerance is set wide, you will risk pulling in incorrect peaks. If the tolerance setting is too narrow, you may not find the right peak. Getting the tolerance levels set correctly may require some trial and error. Try starting with 0.075 Da, a 50 ppm error on a m/z of 1500.
  • Leave the check boxes set to default values.
  • Enter known m/z values for peptide fragment ions. The retention time (RT) will be the same for all fragments and is the average RT for the peptide across all samples. The RT window will depend on the reproducibility of the RT across samples. Set this to accommodate the largest RT shifts observed in the raw data.
  •  Click OK… and wait for samples to process
  • On the results view, check that all target m/z values have been found. Any dots at zero (as pictured below) mean a peak was not able to be found within the specified tolerances. Press Open Samples to restart the process (the same sample will be auto-selected) and adjust tolerances until you find all m/z values.
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  • Use the drop-down menu to check that the other metrics (ppm error, RT) are consistent. A different RT or an inconsistent m/z error could mean an incorrect peak assignment.
  • If all metrics are as expected, start again but add all samples requiring calibration. Repeat the above process.
  • When all samples have been processed and all peaks found, click Recalibrate Samples.
 
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  • Click OK on the warning popup screen.