For research use only. Not for use in diagnostic procedures.
One common error when using the SWATH® Acquisition MicroApp 2.0.1 for processing SWATH data is to relate the extracted ion chromatogram (XIC) width setting in the processing software with the mass accuracy of the instrument, and set the XIC width to a value that is too low. XIC extraction widths should be set to 50 to 75 ppm, regardless of the mass accuracy of the acquired data. The correct value for the XIC width will improve the overall peak intensities, giving better sensitivity (see second image below).
To change the XIC width setting in the SWATH Acquisition MicroApp, either select the Processing Settings button in the general view or browse to the Quantitation menu and select SWATH Processing and then Processing Settings. In the XIC Options section, the user can filter for time width and spectral width.
In the XIC width field, the value should be set to 50 or 75 ppm. Even if the data has a mass accuracy of < 20 ppm, a lower mass accuracy value of 20 ppm should not be used as the XIC width (see image below). When a smaller XIC width is used (20 ppm in red versus 50 ppm in green), the peak area of the 20 ppm XIC is much smaller, which can impact the quantitation of mixed MS/MS spectra of the SWATH data. When the data is centroided, the impact of using a too-small XIC width is even worse, whereby the peak area is completely underestimated (see second chromatogram in image below). Because SWATH peak areas are generally so unpredictable, so its recommend to use a wider XIC width for adequate detection of the low abundance fragment ions.