New features in Mascot 1.5
Scoring Changes
It is no longer necessary to specify which ion series are expected in an MS/MS spectrum.
Mascot now performs internal iteration, testing each ion series independently
in its search for the best match.
You may notice that absolute ions scores are now slightly lower than before.
Previously, the values were too high for a very strong match. This has been corrected.
The algorithm for selecting MS/MS fragment ion peaks on the basis of intensity has
been improved so that it is less sensitive to gross variations in intensity
across the mass scale.
New Peptide Summary Report
The Peptide Summary is a new report that is better suited to MS/MS searches
of either a single peptide or an LC/MS dataset containing a mixture of proteins.
Basically, this is a report driven by matches to peptides, rather than matches
to proteins.
This new report is the default for an MS/MS ions search. Links are
provided near the top of the page to switch between this format and the original
master results report (now called protein summary report).
A significance threshold is reported for each peptide match. Sometimes,
an ions score will be below this threshold, even though the match is clearly
correct. This is because the threshold reflects the probability that the
observed spectrum could have been obtained from the fragmentation of a peptide
of random sequence. However, real spectra also contain noise peaks which are not peptide
related and empty regions which reduce the ions score below that which would
be obtained if the spectrum contained only sequence ion peaks.
Even the best match to a very short peptide can never get a very high score.
The maximum possible score for an ideal match to a 5 residue peptide, for example,
is 65. (Equivalent to 1/20 ^ 5). In a no-enzyme search, any real matches to such short
peptides are likely to be close to or below the significance threshold.
So, if one is looking
at several peptides matches to the same protein, and the total ions score is well
above the threshold for identifying the protein, (as shown in the histogram at the
top of the report), one can still be confident that a top-ranking peptide match is correct,
even though the ions score may be below its significance threshold.
One of the strengths of the Peptide Summary report is that entries that share the same set of
peptide matches are collapsed together into a single hit. If the sample contained a
complex mixture of proteins, the new report provides a clearer and more concise
list of the component proteins.
In the body of the report, when the mouse cursor is placed over a query number
link, a pop-up window displays the top ten peptide matches for that query.
A full decription of both the Peptide Summary and the Protein Summary reports can
be found here.
Variable Modifications
The handling of variable (non-quantitative) modifications has been improved.
If a matched peptide contains a mixture of modified and unmodified sites,
Mascot scores each arrangement independently and reports the best match.
It is now possible to specify multiple variable modifications which target
the same residue. For example, both propionamide and carbamidomethyl as
non-quantitative cysteine modifications.
Remember that the number of calculated peptide masses increases geometrically with the
number of variable modifications. This can dramatically reduce specificity and increase
the time taken to perform a search. Variable modifications should be used intelligently
and sparingly, or the results may be disappointing.
Other Changes
- It is now possible to perform a repeat search following a failed search.
- Peptide view now includes immonium ion masses.
|