This section describes accessing and handling of query-level modifications in Mascot 2.5 and later results files. Query-level modifications were handled as part of the master variable modification list in Mascot 2.4 and earlier, so this information is only useful if you use query-level modifications in Mascot 2.5 or later. See also the section on backwards compatibility.
For simplicity, we will refer to variable modifications specified at search form level or in the quantitation method as form-level modifications, and variable modifications specified in an individual query as query-level or local modifications.
Suppose the search has form-level modifications
IT_MODS=Oxidation (M),Acetyl (N-term), and that there is a peptide match to query 1, which has the query-level modifications
IT_MODS=Phospho (ST). The form-level modification numbers are
1=Oxidation (M) and
2=Acetyl (N-term), while the query-level modifications of query 1 have
To find the modifications of the peptide match, fetch the peptide's variable mods string getVarModsStr() and the query-level mods string getLocalModsStr(). In this case, the variable mods string could be
20100000 and the query-level mods string
00001000. We can decode the strings in two steps:
2010000, look up the modification name using ms_searchparams::getVarModsName(). We find the peptide is acetylated at the N-terminus and oxidised at the second residue.
00001000, look up the modification name using ms_inputquery::getLocalVarModName(). We find the peptide is phosphorylated at the fourth residue.
Points to note:
massessection of the results file, accessible via ms_searchparams, as in Mascot 2.4 and earlier.
IT_MODSparameter in the query section, which can be accessed with ms_inputquery::getIT_MODS(). Parser 2.5 and later parse the value of
IT_MODSand make it available via ms_inputquery::getLocalVarModName(). In Parser 2.4 and earlier, you would need to split the unescaped
IT_MODSmanually to find the query-level modification names.
getLocalModsStr()can be empty in Mascot 2.4 and earlier results files, and it can consist of only zeroes. If you need to test whether a peptide match uses query-level modifications, test that the string is non-empty and contains at least one non-zero digit.
In general, modification number for a residue can be non-zero only in one mods string at a time; that is, a residue cannot have a modification in both the variable mods string and the local mods string. The only exception is when support for multiple modifications on the same site is explicitly enabled, as described in section 3 below.
The neutral loss information of form-level and query-level modifications is encoded in two strings: ms_peptide::getPrimaryNlStr() and ms_peptide::getLocalModsNlStr(). The data format and numbering is analogous to variable modifications.
For form-level mods, use ms_searchparams::getVarModsNeutralLosses() to load the neutral loss vector of the variable modification. If a digit in
getPrimaryNlStr() is non-zero, it refers to an element in the neutral loss vector. The vector is 0-based, so digit 2, for example, is the second element, at index 1.
For query-level mods, you need to locate the modification object first; see getLocalModsStr() for an example workflow. The correct neutral loss vector can then be loaded with ms_modification::getNeutralLoss(). As above, the non-zero digits in
getLocalModsNlStr() refer to elements in the neutral loss vector.
Mascot 2.4 added support for Multiple modifications for the same site in certain quantitation configurations, which is triggered when the search is run with the
MULTI_SITE_MODS=1 parameter. In this mode,
getVarModsStr() describes the exclusive modifications defined in the quantitation method, and getSummedModsStr() describes variable modifications occuring at the same sites as the exclusive modifications.
For example, suppose you have a results file using the SILAC quantitation method, which specifies
Label:13C(6)15N(2) (K) as an exclusive modification. Suppose you also specify variable modification
Acetyl (K) in the search form, so that the form-level modifications are
1=Acetyl (K) and
2=Label:13C(6)15N(2) (K). When
MULTI_SITE_MODS=1, a peptide match could have
getVarModsStr() == 00020000 and
getSummedModsStr() == 00010000 That is, the former contains the (exclusive) SILAC label on K and the latter the variable modification (acetylation) on the same residue.
Now suppose the input query has also the query-level modifications
IT_MODS=Methyl (KR), so that
1=Methyl (KR). Peptide matches to the query could have the following combinations of mods strings:
|Rank 1||Rank 2||Rank 3|
In this case, the rank 1 match has acetylation and SILAC label on K (residue 3), while the rank 2 match has methylation and SILAC label on K (residue 3). The rank 3 match has acetylation and SILAC label on K (residue 3), and methylation on K or R at the C terminus (residue 6). Observe that in the rank 2 match, the summed mods string is empty and both the variable mods string and the local mods string contain a non-zero value for the third residue. This is the only case where
getLocalModsStr() can report a non-zero digit at the same site.
As a mnemonic, the summed and local modifications are always in addition to ("summed with") the exclusive modifications, which are recorded in
The peptide match object can have query-level modifications (a non-empty
getLocalModsStr()). In this case, the query-level modifications are only included in fragment mass calculation if the peptide object is directly related to the results file with which the
ms_aahelper object was initialised. That is, to calculate the right fragments for peptide matches with query-level modifications:
If the peptide object comes from a different file or if you initialise
ms_aahelper without a results file, fragmentation will not take query-level modifications into account and some of the fragment masses will be incorrect.
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