If you obtain unexpected quantification results, verify that all node settings in your processing workflow are reasonable.

  • Make sure that the Integration Tolerance parameter of the Reporter Ions Quantifier node fits the data that you are processing. The default is 20 ppm, which is too low if you are processing PQD data from an ion trap.
  • Make sure that the settings of the Mass Analyzer, MS Order, and Activation Type parameters of the Reporter Ions Quantifier node are correct for the data that you are processing. The following figure shows the typical settings to use for quantifying HCD scans from the Orbitrap mass spectrometer.
Typical settings for quantifying iTRAQ or iodo TMT 6plex tags from HCD scans
Typical settings for quantifying iTRAQ or iodo TMT 6plex tags from HCD scans

To quantify PQD scans from an ion trap, use the typical settings.

Typical settings for quantifying ITRAQ or TMT tags from ion trap PQD scans
Typical settings for quantifying ITRAQ or TMT tags from ion trap PQD scans

Make sure that you use the correct set of static and dynamic modifications for the search engine. For example, if you are searching iodo TMT 6plex data with Sequest HT, check that your settings resemble those in the following figure.

Modifications required for searching iodo TMT 6plex samples
Modifications required for searching iodo TMT 6plex samples