The Proteome Discoverer application includes an algorithm for mass recalibration. This algorithm recalculates precursor mass independently per file. It calculates a correction curve by using support vector regression. The Mass Recalibration view indicates how well the regression performed. It can also provide insight into potential issues with the acquisition or chromatography itself. It displays the input data (called landmarks in this context) used for the regression and the calculated correction curve.

For mass recalibration, perform a fast Sequest HT search with a fairly wide precursor mass tolerance (2 ppm by default) to identify enough confident identifications to deduce the mass error from. You must use the Spectrum Files RC node instead of the Spectrum Files node in the processing workflow to generate a result file that can display the Mass Recalibration view.

For more information, see Incorporating Mass Recalibration. For information on the Mass Recalibrations page of the result file, see The Mass Recalibrations page.

Procedure

  1. In the result file, select the Input Files or the Mass Recalibrations tab (use the Result Table Chooser to make these tabs visible if necessary).
  2. The Input Files page displays the input data for the regression and the calculated correction curve.
  3. The Mass Recalibration page indicates how well the recalibration performed for all the input files.
  4. Select an input file on the Input Files page or the Mass Recalibration page.
  5. Select View > Mass Recalibration View.
  6. The Mass Recalibration view opens.
  7. Mass Recalibration view

The Mass Recalibration view displays the distribution of detected mass errors over the retention time range. It also shows you how the application fit the correction curve to the data.

  • The x-axis value used for the regression is the mass error between the measured mass for a given peptide and the theoretical mass based on the identified sequence, in parts per million.
  • The y-axis value is the deviation of the experimental mass of the peptide match from the theoretical mass of the peptide.