Description, Instructions, and Tips for


This document provides instructions for .

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was originally developed for use with those MALDI-PSD spectra which do not contain a sufficient number of peaks to enable de novo sequence interpretation, but were obtained from peptides whose sequence might be present in a database. The program can also be used with data from MS/MS techniques other than MALDI-PSD simply by selecting fragment-ion types consistent with the technique. MS-Tag can also be used with spectra which contain sufficient information content to enable de novo interpretation; however, a homolog of the peptide must be in the database for a search to be successful.

Fragment-Ion types

Checking the boxes next to each ion type allows those ion types to be considered in matching the data to a sequence. Users can either choose to use the default ion types for a particular instrument or a user selected set. Server administrators can modify the default instrument ion types or add new instrument selections. The instrument definition can also include a specification of which amino acids lose water (default S,T,E and D), which lose ammonia (default R,K,N and Q), which are positive charge bearing (default R,H,K and N) and what the maximum internal ion mass is (default 700 Da). Selection of fewer ion types will generally lead to fewer false positives, however ion masses corresponding to ion types not selected or unknown to the program will result in those ion masses NOT being matched. The -SOCH4 and -H3PO4 ion types should only be used in homology mode, and should be selected when the spectrum also contains MH+ - 64 and MH+ - 98 ions respectively. The currently supported ion types are:

Ion type Restrictions
a, b, y no restrictions
c not if C term residue of the fragment is P
a-NH3, b-NH3, y-NH3 ion contains an amino acid which loses ammonia
b-H2O ion contains an amino acid which loses water
b+H2O ion contains a positive charge bearing amino acid; only bn-1, bn-2 ( length n)
a-H3PO4, b-H3PO4, y-H3PO4 ion contains phosphorylated S,T
b-SOCH4, y-SOCH4 ion contains oxidized M
internal b < maximum internal mass
internal a < maximum internal mass, internal b present
internal b-H2O < maximum internal mass, internal b present, ion contains an amino acid which loses water
internal b-NH3 < maximum internal mass, ion contains contains an amino acid which loses ammonia
N-term ladder removal of N term residues (y equiv.)
C-term ladder removal of C term residues (b+H2O equiv.)

Mass Tolerances

While the intact MW of the protein can be specified, MS/MS data is generally of such high discriminating power that no restriction is necessary. Furthermore, database entries frequently contain pre and pre-pro proteins, as well as gene fragments.
The tolerances on both the parent ion and fragment ions should be set to be consistent with the mass accuracy of the instrument used to generate the data. It is generally a better idea to use units of ppm or % rather than Da. Fragment mass accuracy is often better at lower mass than at higher mass. For PSD spectra this is particularly important. If you have +/- 1 Da tolerance you are more likely to match low mass internal sequences of the wrong nominal mass thus increasing the likelihood of false-positive hits. Hence setting the fragment mass accuracy to +/- 1000 ppm prevents this with an "acceptable" trade-off of allowing looser than appropriate mass accuracy on fragment ions > 1000 Da.

What is a Fragment-ion Tag?

A Fragment-Ion Tag can be obtained from an MS/MS spectrum and consists of 3 attributes:

Fragment-ion Tag
  1. A peptide parent-ion mass: Pm.
  2. Masses of all sequence related fragment-ions from the peptide: Fi, Fj, ... The fragment ions need not all be of the same ion type. Currently supported fragment-ion types are described above.
  3. Masses of composition ions which indicate the presence of particular amino acids in the peptide: Ci, Cj, ... This information can be from immonium and related low mass ions or high mass ions representing side-chain losses from the parent ion. If the amino acid compositional ions are entered either by checking the appropriate box or by including the ions in the fragment ion mass list, the presence of the corresponding amino acid will be a requirement for a successful match. For example, if 110 is entered into the mass list, the database will be screened to include only H containing peptides. These compositional ions are all less than 186. The screening is done in all versions of MS-TAG. When in doubt, leave it out!