If the retention times (RT) within the ion library to be used differ from the retention times that will be observed in the SWATH acquisition data to be processed, it is important to recalibrate the retention times within the ion library.

If a Retention Time Calibration Protein already exists within your selected ion library, select the Use RT Calibration protein option.

If there is no Retention Time Calibration Protein in your ion library, then you can select the Use Auto-calibration option and a RT Calibration protein will automatically built. In this case, Extractor will automatically build a RT Calibration Protein using endogenous peptides. First, it splits the library time range into 100 equal time bins. In each time bin it selects the best peptide based on precursor intensity and then ID confidence. If there happens to be no peptides in a time bin, it selects from a neighboring time bin until 100 compounds are found. Next it extracts the peak groups for all peptides and computes the peak group score. Only the peptides with scores above the median score for the set of peptides will be used for RT calibration.

In both cases, Extractor will automatically use these pre-selected peptides and detect them in your sample to correct for RT variation between current sample and ion library. To ensure that these are found a wider RT window is used at this point. Once found, a linear transformation will done between the RT where the peptides were detected vs the expected retention time in the library, This allows the RTs in the ion library to be corrected to the retention time frame of the current data files. Then all peptides are extracted using the XIC extraction window that you have set. This retention calibration is done on a file by file basis.

RUO-MKT-18-6401-A