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Adding Custom Modifications

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Robert Millikin edited this page Oct 31, 2019 · 18 revisions

Overview

There are two methods to add a new modification to MetaMorpheus: via the graphical user interface (GUI) or by editing MetaMorpheus's text files. The first method is recommended because there is error-checking that takes place in the GUI that prevents most mistakes. Editing the text file is error-prone and can cause MetaMorpheus to crash on startup. However, advanced users who are adding many complicated modifications may wish to opt for the text-editing route to save time.

Modification properties

  • ID - This is the modification's name.
  • Modification type - This specifies which category the modification belongs to. Some existing modification types include "Common Biological", "Common Artifact", etc. The user is free to designate their own type, which creates a separate list.
  • Motif target - Amino acid letter code, capitalized. The capital letter 'X' may be used to mean any amino acid. Multiple-letter motifs are allowed, but only one amino acid is the "target" amino acid and should be capitalized. For example, "Ykk" would place the modification on any tyrosine residue that is followed by two lysines.
  • Chemical formula - This is the chemical formula of the added or removed atoms. Either the chemical formula or the modification's monoisotopic mass must be specified. A particular isotope of the element can be specified in curly braces following the element name. For example, carbon-13 is written as C{13}. The number of atoms is specified after the closing brace. Five carbon-13 atoms is written as C{13}5.
  • Monoisotopic mass - The mass shift produced by the modification. Use at least 5 decimal places. This will override the chemical formula. This is because there are cases where the mass of the mod and the mass shift from the mod are different (e.g. trimethylation has mass of 43 Da but mass shift from trimethylation is 42 Da).
  • Position - Choose from the following options: Anywhere.; Peptide N-terminal.; N-terminal.; Peptide C-terminal.
  • Neutral loss - Certain PTMs (e.g. phosphorylation) have labile modifications that can be lost during fragmentation. Specifying neutral loss tells MetaMorpheus to look for fragments with a neutral loss.
  • Diagnostic ions - Certain PTMs (e.g. acetylation or glycosylations) produce diagnostic fragment ions that can be detected in MS/MS spectra. These ions can serve as useful indicators of the presence of the corresponding PTM. These ions are searched and, if found, annotated.

Adding a modification through the GUI

  • Pros: Less complicated than editing a text file; less prone to errors
  • Cons: Can only specify one motif for a modification; can only specify one dissociation type for neutral loss and diagnostic ions; not available to command-line users

In MetaMorpheus, go to Data -> Create new modification. The required items are marked with a red asterisk. Essentially, you must give the modification a name ("ID"), a category ("Modification Type"), an amino acid position or motif ("Motif"), a monoisotopic mass or chemical formula, and a general location (peptide or protein N-terminus, peptide or protein C-terminus, or anywhere along the amino acid chain, subject to the motif requirements). Optionally, you can specify neutral loss(es) and diagnostic ions that occur upon fragmentation with the specified dissociation type.

Adding a modification via editing text files

  • Pros: All functionality of MetaMorpheus's modification system is available
  • Cons: Complicated and error-prone

The existing modifications are organized in text files in the "Mods" folder of MetaMorpheus. This is best accessed directly through the MetaMorpheus GUI application by selecting Data -> Open folder with mods/data files -> Mods. You can access the Mods folder directly in Windows using the File Explorer in the \User***\AppData\Local\MetaMorpheus\Mods folder. Command-line users will find a mods folder in the MetaMorpheus root directory. The user is free to add modifications by either editing those files or by creating a new text file (preferably using Notepad++, which helps get the syntax correct). It is recommended to add a new text file in the Mods directory rather than editing an existing file; the files containing MetaMorpheus's modifications WILL BE OVERWRITTEN during software updates unless the modifications are added to a new text file (e.g. user_custom_mods.txt).

Each modification contains a number of fields designated by a two letter code (see list below). Each two-letter code must be followed by three spaces, then the information for the field. Each key-value pair must be on a separate line.

  • AC Accession An accession number of frequently supplied by the primary databases (e.g. UniProt and Unimod).

  • CF Chemical formula (required if no MM is supplied/defined) This is the chemical formula of the added or removed atoms. This is required but the mass shift used is specified by MM. The particular isotope of the element can be specified in curly braces following the element name. For example, carbon-13 is written as C{13} in the chemical formula. The number of atoms is specified after the closing brace. Five carbon-13 atoms is written as C{13}5.

  • DI Diagnostic Ions Certain PTMs (e.g. acetylation or glycosylation) produce small diagnostic fragment ions that can be detected in MS/MS spectra. These ions can serve as useful indicators of the presence of the corresponding PTM.

  • DR External database links

  • FT Feature key Used in the UniProt ptmlist but not needed for custom mods in MetaMorpheus

  • ID Identifier (Required) This is the text used to describe the modification in the output.

  • MM Monoisotopic mass (Required if CF is not supplied/defined) The exact atomic mass shift produced by the modification. Please use at least 5 decimal places of accuracy. This will override the monoisotopic mass described in the chemical formula because there are cases where the mass of the mod and the mass shift from the mod are different (e.g. trimethylation has mass of 43 but mass shift from trimethylation is 42).

  • MT Modification type (Required) This specifies which modification group the modification should be included with. Existing modification types are described here. The user is free to designate their own type, which creates a separate list.

  • NL Neutral loss (if any) Certain PTMs (e.g. phosphorylation) have labile modifications that can be lost during fragmentation. Specifying neutral loss tells MetaMorpheus to look for fragments with a neutral loss.

  • PP Position of the modification in the polypeptide (Required) Choose from the following options: Anywhere.; Peptide N-terminal.; N-terminal.; Peptide C-terminal. DON'T FORGET THE '.'

  • TG Target (Required) Amino acid letter code capitalized or written out. Multiple targets separated by " or ". The capital letter 'X' may be used to mean any amino acid.

  • // Break between modifications

Examples

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