Matches with same score are ambiguous

Mascot may give the same score to two or more matches in the same query. A typical case is an I/L difference in peptide sequences, say CIPALDSLTPANEDQK and CIPALDSITPANEDQK. Because leucine (L) and isoleucine (I) have exactly the same molecular weight, the calculated fragments for these two peptides are also exactly the same. The two matches get the same score when matched against the same spectrum.

Parser signals this ambiguity by pretty rank, accessible with ms_peptide::getPrettyRank(). Two peptides with the same score have the same pretty rank. For example, if CIPALDSLTPANEDQK and CIPALDSITPANEDQK are the rank 1 and rank 2 matches, both have pretty rank 1.

Note that score-level ambiguity has little to do with ms_peptide::getAmbiguityString(), which encodes residue substitutions based on ambiguous residues in protein sequences.

Ambiguity group IDs in integrated spectral library searches

Mascot 2.6 and later support Spectral libraries. Integrated spectral library searches may have ambiguity of a different kind. Within a query, two peptide matches are ambiguous if they have the same pretty rank (as above) or they have the same sequence but come from a different search engine. Ambiguous sequences are automatically taken into account during protein inference.

Consider the following matches to a query:

Rank	Sequence	Score	Source	Pretty rank	Ambiguity group
1	CIPALDSLTPANEDQK	524	SL	1	1
2	CIPALDSLTPANEDQK	399	SL	2	2
3	CIPALDSLTPANEDQK	51	AA	3	1
4	CIPALDSITPANEDQK	51	AA	3	1
5	CIPALDSLTPANEDQK	310	SL	4	3
6	SLNNQIETLLTPEGSR	250	SL	5	4

The rank 1 match starts ambiguity group numbering, exactly the same as in the pretty rank case.

The rank 2 match is in a different ambiguity group than rank 1. Although the sequences are the same, the matches have the same search engine (both from spectral library) but different score (they probably had different library spectra).

Rank 3 and 4 matches have the same pretty rank; they are the typical I/L case. Because the rank 3 match has the same sequence and different search engine than the rank 1 match, ranks 1, 3 and 4 belong to the same ambiguity group.

The rank 5 match is again in an ambiguity group of its own for the same reason as the rank 2 match.

Finally, the rank 6 match has a different sequence and different score from the rest, so it also is in its own ambiguity group.

Ambiguity grouping for a query is available with ms_peptidesummary::getAmbiguityRanks().