Mascot: The trusted reference standard for protein identification by mass spectrometry for 25 years

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 description 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.