If you obtain unexpected precursor ion quantification results, verify that all settings of your processing workflow are reasonable.
- Check the dynamic modification parameters in the Mascot or Sequest HT search engines. Ensure that they match your isotope labeling sample.
- Check the node parameters that you set before performing the quantification to see if they are appropriate for your sample.
For more information, see Creating Precursor Ion Quantification Workflows. - Verify that your isotopic labeling is one of the following options in the protein ID/search node (either Sequest HT or Mascot):
- SILAC 2plex (Arg10, Lys6): Uses arginine 10 and lysine 6.
- SILAC 2plex (Arg10, Lys8): Uses arginine 10 and lysine 8.
- SILAC 2plex (Ile6): Uses isoleucine 6.
- SILAC 3plex (Arg6, Lys4|Arg10, Lys8): Uses arginine 10 and lysine 8 for “heavy” labels and arginine 6 and lysine 4 for “medium” labels.
- SILAC 3plex (Arg6, Lys6|Arg10, Lys8): Uses arginine 10 and lysine 8 for “heavy” labels and arginine 6 and lysine 6 for “medium” labels.
- Dimethylation 3plex: Chemically adds isotopically labeled dimethyl groups to the N-terminus and to the e-amino group of lysine.
- 18O labeling: Introduces 2 or 4 Da mass tags through the enzyme-catalyzed exchange reaction of C-terminal oxygen atoms with 18O.
NOTE
Low-mass accuracy MS 1 full-scan data cannot be used for precursor ion quantification.
- Check your tolerance window. If you get too many results, decrease the size of the window. For too few results, increase the size of the window.
- Make sure you chose the right database.
- Check the species listed to make sure the samples came from that species.
- Verify that the activation type used is correct.
- Verify that the instrument type in the Mascot search engine is correct.
- Use only the ETD Spectrum Charger node for low-mass resolution ETD data.