Comet parameter: variable_mod01 through variable_mod09

  • There are 7 entries/settings that are associated with these parameters:
    • The first entry is a decimal value specifying the modification mass difference.
    • The second entry is the residue(s) that the modifications are possibly applied to. If more than a single residue is modified by the same mass difference, list them all as a string. Use 'n' for N-terminal modfication and 'c' for C-terminal modification.
    • The third entry is a integer to specify whether the modification is a variable modification (0) or a binary modification (non-zero value).
      • 0 = variable modification analyzes all permutations of modified and unmodified residues.
      • non-zero value = A binary modification analyzes peptides where all residues are either modified or all residues are not modified.
      Note that if you set the same binary modification value in multiple "variable_mod?" parameter entries, Comet will treat those variable modifications as a "binary set". This means that all modifiable residues in the "binary set" must be unmodified or modified. Multiple "binary sets" can be specified by setting a different binary modification value e.g. use "1" for all modifications in set 1, and "2" or all modifications in set 2. Binary groups were added with version 2015.02 rev. 1.
    • The fourth entry is an integer specifying the maximum number of modified residues possible in a peptide for this modification entry.
    • The fifth entry specifies the distance the modification is applied to from the respective terminus:
      • -1 = no distance contraint
      • 0 = only applies to terminal residue
      • 1 = only applies to terminal residue and next residue
      • 2 = only applies to terminal residue through next 2 residues
      • N = only applies to terminal residue through next N residues where N is a positive integer
    • The sixth entry specifies which terminus the distance constraint is applied to:
      • 0 = protein N-terminus
      • 1 = protein C-terminus
      • 2 = peptide N-terminus
      • 3 = peptide C-terminus
    • The seventh entry specifies whether peptides are must contain this modification. If set to 1, only peptides that contain this modification will be analyzed.
      • 0 = not forced to be present
      • 1 = modification is required
  • Modification codes for variable_mod01 through variable_mod09 (for some outputs): *, #, @, ^, ~, $, %, !, +
  • The default value is "15.9949 M 0 3 -1 0 0" if this parameter is missing for variable_mod01.
  • The default value is "0.0 X 0 3 -1 0 0" if this parameter is missing for variable_mod02 through variable_mod09.

Example:
variable_mod01 = 15.9949 M 0 3 -1 0 0
variable_mod02 = 79.966331 STY 0 3 -1 0 0     ... possible phosphorylation on any S, T, Y residue
variable_mod02 = 79.966331 STY 0 3 -1 0 1     ... force peptide IDs to contain at least one phosphorylation mod
variable_mod01 = 42.010565 nK 0 3 -1 0 0     ... acetylation mod to lysine and N-terminus of all peptides
variable_mod01 = 15.994915 n 0 3 0 0 0     ... oxidation of protein's N-terminus
variable_mod01 = 28.0 c 0 3 8 1 0     ... modification applied to C-terminus as long as the C-term residue is one of last 9 residues in protein
variable_mod03 = -17.026549 Q 0 1 0 2 0     ... cyclization of N-terminal glutamine to form pyroglutamic acid (elimination of NH3)
variable_mod04 = -18.010565 E 0 1 0 2 0     ... cyclization of N-terminal glutamic acid to form pyroglutamic acid (elimination of H2O)

Here is a binary modification search example of triple SILAC plus acetylation of lysine. The SILAC modifications are "R +6 and K +4" (medium) and "R +10 and K +8" (heavy). In conjunction with K +42 acetylation, the binary modification sets would be "R +6, K +4, K +4+42" for SILAC medium (binary group 1) and "R +10, K +8, K +8+42" for SILAC heavy (binary group 2). Mass values are listed with no precision for clarity; definitely use precise modification masses in practice.
variable_mod01 = 42.0 K 0 3 -1 0 0
variable_mod02 =  6.0 R 1 3 -1 0 0
variable_mod03 =  4.0 K 1 3 -1 0 0
variable_mod04 = 46.0 K 1 3 -1 0 0
variable_mod05 = 10.0 R 2 3 -1 0 0
variable_mod06 =  8.0 K 2 3 -1 0 0
variable_mod07 = 50.0 K 2 3 -1 0 0
variable_mod08 =  0.0 X 0 3 -1 0 0
variable_mod09 =  0.0 X 0 3 -1 0 0