For reporter ion quantification, the Quantification Spectrum chart displays the intensity of the reporter ions, in counts. It shows a spectrum for each peptide, except for those peptides labeled with “No Quan Values” in the Quan Info column of the PSMs page.

Procedure

  1. Select the PSMs, Quan Spectra, or MS/MS Spectrum Info tab in the result file.
  2. Select the row of the quantification spectrum that interests you.
  3. Choose View > Quantification Spectrum, or select Quantification Spectrum .
  4. The following figure shows an example of a quantification spectrum from a TMT 10plex sample.
  5. Quantification Spectrum chart for a TMT 10plex showing the intensity of reporter ions
  6. The width of the bars shown in the Quantification Spectrum chart is determined by the setting of the Integration Tolerance parameter in the Reporter Ions Quantifier node in the processing workflow.
  7. The Quantification Spectrum chart for reporter ion quantification includes the following features:
  8. The light blue bars represent the integration windows for the reporter tags. The boxes are centered on the masses of the reporter tags, as specified in the quantification method. The width of boxes is the integration window used for extracting the reporter tags. It is ±20 ppm, as specified by the settings of the parameters in the Reporter Ions Quantifier node in the processing workflow (you can look up all these values on the Quantification Summary page). The height of the line in the box represents the actual tag intensity used for calculating the peptide ratios. The height of the bars represents the corrected tag intensity. The height depends on the setting of the Integration Method parameter specified in the Reporter Ions Quantifier node in the processing workflow. It is always the value that results from correction for isotopic impurities, as specified in the TMT Reporter Ion Isotope Distributions area of the Quan Channels page of the Quantification Method Editor dialog box.
  9. The bright blue lines represent the peaks used for reporter integration. To calculate the actual intensity of a particular tag, the application chooses the blue fragment peaks from the spectrum and considers only peaks in the integration window.
  10. The pink bars denote the theoretical quantification windows that indicate where the peaks should be.
  11. Theoretical integration windows marked by pink bars in the Quantification Spectrum chart
  12. The black fragment peaks represent peaks that are present in the spectrum but that are not chosen for calculating the tag intensities. They might not be chosen because the peaks lie outside of any integration window, or because the setting of the Integration Method parameter specified in the Reporter Ions Quantifier node determined that only one peak per integration window should be chosen from any integration window. A different peak was picked for this integration window according to the criterion specified by the Integration Method setting.