Here's some notes on setting Comet search parameters for various isotopic/isobaric labeling strategies.

• ITRAQ 4-plex

  The 4-plex reagent has different monoisotopic mass values for 114 (144.105918), 115 (144.09599), and 116/117 (144.102063). The mass value used below is derived from averaging the three monoisotopic masses. The "clear_mz_range" parameter is used to remove any reporter ion signal from the spectra so that they aren't matched as fragment ions.

  add_Nterm_peptide = 144.10253
  add_K_lysine = 144.10253
  clear_mz_range = 113.5 117.5

• ITRAQ 8-plex:

  Similarly, the 8-plex reagent has two different set of masses for 115/118/119/121 (304.199040) and 113/114/116/117 (304.205360). The mass modification below is the average of the two.

  add_Nterm_peptide = 304.2022
  add_K_lysine = 304.2022
  clear_mz_range = 112.5 121.5

• TMT 2-plex:

  add_Nterm_peptide = 225.155833
  add_K_lysine = 225.155833
  clear_mz_range = 125.5 127.5

• TMT 6-plex and 10-plex:

  add_Nterm_peptide = 229.162932
  add_K_lysine = 229.162932
  clear_mz_range = 125.5 131.5

• SILAC 4 Da:

  There are a number of different SILAC reagents with a ~4 Da modification (based on combinations of C13 and N15), each with different sites of specificity. My example below is for the 15N(4) reagent applied to R residues. Adjust the modification mass and residue(s) applied to as necessary.

  To perform a mixed light/heavy search using a variable modification search in binary mode (binary mode = no mixing light and heavy modifications within a peptide so all arginine residues are considered light or all arginine residues are considered light):

  variable_mod01 = 3.988140 R 1 3 -1 0 0

  To search just the heavy labeled sample, you can apply a static modification:

  add_R_lysine = 3.988140

• SILAC 6 Da:

  I'm using the 13C(6) SILAC mass in the example below assuming it's applied to both K and R; adjust as necessary. There's at least one more SILAC reagent with ~6 Da modification mass and different residue specificity: 13C(5) 15N(1)

  To perform a mixed light/heavy search using a variable modification search in binary mode (binary mode = no mixing light and heavy modifications within a peptide so all arginine+lysine residues are considered light or all arginine+lysine residues are considered light):

  variable_mod01 = 6.020129 KR 1 3 -1 0 0

  To search just the heavy labeled sample, you can apply a static modification:

  add_K_lysine = 6.020129
  add_R_arginine = 6.020129

• SILAC 8 Da:

  The example below is for 13C(6) 15N(2) on K residues.

  Variable (binary) modification search:

  variable_mod01 = 8.014199 K 1 3 -1 0 0

  Static modification for just the heavy labeled search:

  add_K_lysine = 8.014199