How does the mass recalibration work in ProteinPilot software?
To perform automatic mass recalibration, the software first performs a Rapid search using mass tolerances that are wider than what is expected for the instrument used. The mass tolerance used in this initial search is defined in the MSTOLERANCE VALUE and MSMSTOLERANCE VALUE setting in the parameter translation file (Figure 1, refer to the parameter translation file to see what these values are for your instrument). The high confidence identifications (non-modified tryptic peptides) from this quick search can then be used to determine the mass shift and compute a mass correction. This is actually performed in segments across the duration of the LC run, just in case there is some minor drift across the run time (binned by every 4000 MS/MS spectra). Each segment is individually recalibrated, both the MS and the MS/MS data. To compute the calibration within each RT range, the m/z range is binned and the distribution of the delta between theoretical and observed m/z in each bin is evaluated for the mode location. Pre-defined m/z tolerances around mode of the delta in each m/z bin are used to reject outliers from the analysis. A linear regression is done in TOF (sqrt(m/z)) domain
Now we have a recalibrated peak list, this can be used in the final search, using the tighter mass accuracy settings appropriate for a well-calibrated instrument of your instrument type. These tolerances are defined in the parameter translation file as the MS_STANDARD_DEVIATION VALUE and MSMS_STANDARD_DEVIATION VALUE (Figure 1) and reflect the typical mass variation expected for each instrument. The final mass errors that are reported are based on the recalibrated results.
The calibration is done separately for MS and MS/MS experiments.
In ProteinPilot software 5.0, you can now export this recalibrated peak list as a *.mgf file, if you want to compare results to other search engines.
Interested in investigating the impact of mass recalibration? (For advanced users)
Although we recommend using mass recalibration at all times, the algorithm can be turned off you want to investigate its impact on your results.
To do so:
- Close ProteinPilot software and open the parameter translation file and edit as described below to turn mass recalibration off.
- Find the instrument definition for your TripleTOF system type, find the AUTO_CALIBRATION flag and change the True to False to turn off recalibration.
- Remember to turn it back on by repeating steps 1 and 2 (changing False to True) after you have performed your search!
C:\Program Files\AB SCIEX\ProteinPilot\WorkflowDirectory\ParameterTranslation.xml