Comet parameter: variable_mod01 through variable_mod09

There are 8 fields/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). 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 modification groups were added with version 2015.02 rev. 1.
  • 0 = variable modification analyzes all permutations of modified and unmodified residues.
  • non-zero value = binary modification analyzes peptides where all residues are either modified or all residues are not modified.
• The fourth entry is an integer specifying the maximum number of modified residues possible in a peptide for this modification entry. With release 2020.01 rev. 3, this field has been extended to allow specifying both a minimum and maximum number of modified residues for this modification entry. A single integer, e.g. “3”, would specify that up to 3 variable mods are allowed. Comma separated values, e.g. “2,4” would specify that peptides must have between 2 and 4 of this variable modification.
• The fifth entry specifies the distance the modification is applied to from the respective terminus:
  • -2 = apply anywhere except c-terminal residue of peptide
  • -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 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
• The eigth entry is an optional fragment neutral loss field. For any fragment ion that contain the variable modification, a neutral loss will also be analyzed if the specified neutral loss value is not zero (0.0).
• The default value is “0.0 X 0 3 -1 0 0 0.0” if this parameter is missing except if Comet is compiled with the Crux flag on. For Crux compilation, the default value for variable_mod01 is “15.9949 M 0 3 -1 0 0 0.0” if this parameter is missing.

Example:

variable_mod01 = 15.9949 M 0 3 -1 0 0 0.0
variable_mod02 = 79.966331 STY 0 3 -1 0 0 97.976896  ... possible phosphorylation on any S, T, Y residue with a neutral loss of 98
variable_mod02 = 79.966331 STY 0 3 -1 0 1 0.0        ... force peptide IDs to contain at least one phosphorylation mod
variable_mod01 = 42.010565 nK 0 3 -1 0 0 0.0         ... acetylation mod to lysine and N-terminus of all peptides
variable_mod01 = 15.994915 n 0 3 0 0 0 0.0           ... oxidation of protein's N-terminus
variable_mod01 = 28.0 c 0 3 8 1 0 0.0                ... 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 0.0          ... cyclization of N-terminal glutamine to form pyroglutamic acid (elimination of NH3)
variable_mod04 = -18.010565 E 0 1 0 2 0 0.0          ... 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 0.0
variable_mod02 =  6.0 R 1 3 -1 0 0 0.0
variable_mod03 =  4.0 K 1 3 -1 0 0 0.0
variable_mod04 = 46.0 K 1 3 -1 0 0 0.0
variable_mod05 = 10.0 R 2 3 -1 0 0 0.0
variable_mod06 =  8.0 K 2 3 -1 0 0 0.0
variable_mod07 = 50.0 K 2 3 -1 0 0 0.0
variable_mod08 =  0.0 X 0 3 -1 0 0 0.0
variable_mod09 =  0.0 X 0 3 -1 0 0 0.0