Matrix Science
Home Mascot Help  
   
  Help > MS/MS Ions Search   
 
 
 
Related Topics
Search parameter reference
Data file format
Result Report Overview
MS/MS Results Interpretation
Summary Reports for MS/MS
Error tolerant search
Decoy Databases
Quantitation
 

MS/MS Ions Search

The MS/MS ions search accepts data in a number of different peak list formats. A single MS/MS spectrum may be searched, or a multi-dimensional LC-MS/MS run containing data from many thousands of peptides.

Search Parameter Notes

Database

If you are searching a single organism database, or using a taxonomy filter, its a good idea to include a contaminants database in the search. You can never guarantee the absence of contaminant proteins, such as the enzyme used for the digest, BSA, keratins, etc. If you don't include a contaminants database, then the result report will show matches to similar proteins from your organism of interest, which may be misleading.

Taxonomy

If you want to use a taxonomy filter for a category that is not listed on the default list, you can easily configure this. Refer to Chapter 9 of the Setup & Installation manual. Categories can be any organism with a TaxID and can easily be combinations of organisms, e.g. human+mouse.

Enzyme

An enzyme of low specificity, which digests proteins to a mixture of very short peptides, is not a good choice, because almost any given 3 or 4 residue peptide will be found in many database entries. The longer the peptide, the greater the specificity. In most cases, it is best to use an enzyme of specificity equal to or greater than trypsin.

Setting the number of allowed missed cleavage sites to zero simulates a limit digest. If you are confident that your digest is perfect, with no partial fragments present, this will give maximum discrimination and the highest score. If experience shows that your digest mixtures usually include some partials, that is, peptides with missed cleavage sites, you should choose a setting of 1, or maybe 2 missed cleavage sites. Don't specify a higher number without good reason, because each additional level of missed cleavages increases the number of calculated peptide masses to be matched against the experimental data. If the actual digest does not contain extended partials, this simply increases the number of random matches, and so reduces discrimination.

The high level of specificity of an MS/MS ions search means that it is not essential to choose an enzyme. However, a search in which an enzyme has been specified will be considerably faster and more sensitive. This is because the "no enzyme" search must test all possible subsequences of each protein, rather than just (say) tryptic peptides. For a protein of N residues, there are approximately N/10 tryptic peptides compared with N(N+1)/2 possible subsequences. For a modest size protein of 250 residues, this is an increase of three orders of magnitude in the number of peptides which must be considered.

Sometimes, you have no choice but to use no enzyme. For example, when the peptides do not originate from a formal digest, such as endogenous peptides. In all cases where the sample has been digested with a known enzyme, it is advisable to specify that enzyme for the search, even though there may be some non-specific cleavage products. There are two ways to pick up non-specific cleavage. If there is a very high level of non-specific cleavage, use a semi-specific version of the enzyme. This will match peptides that are non-specific at one terminus, which minimises the increase in the search space and avoids too great a loss of sensitivity. For typical levels of non-specificity (< 5%) an error tolerant search is the preferred option. This is a second pass search combining a semi-specific version of the selected enzyme plus a search for unusual modifications.

Modifications

For a fast and sensitive search, use a minimum of variable modifications. An error tolerant search is a much better way to find rare modifications than selecting them as variable modifications.

You cannot select two fixed modifications with the same specificity. If you select variable modifications with the same specificity as a fixed modification, this excludes the possibility of an unmodified site. For example, if you choose Carbamidomethyl (C) as fixed and Propionamide (C) as variable, you can get matches to either of these but never to a peptide with free cysteine. Also, you will not get matches to peptides containing both carbamidomethyl and propionamide.

Mass Tolerances

In an MS/MS search, the peptide mass tolerance determines the number of candidate peptides tested for a match. This affects the significance threshold score, but it does not affect the ions score. The match is the same match whether there was 1 candiate or 10,000 candidates. The number of candidates simply determines whether the match is significant or not.

Sometimes, peak detection chooses the 13C peak rather than the 12C. In extreme cases, it may pick the 13C2 peak. If this is happening, use the #13C setting to match these peaks without using a very large peptide mass tolerance.

There is a common misunderstanding that ions scores should continue to increase indefinitely as fragment mass accuracy improves. This is not the case because we are not simply scoring the quality of the spectrum, we are scoring whether the reported match could occur by chance. Scores for good matches approach an asymptotic limit as the fragment mass tolerance approaches zero. In the limit, with perfect mass accuracy and infinite S/N, the Mascot score for a spectrum with complete sequence coverage should approach the score for an identity match in a Blast search, because the MS/MS spectrum can only correspond to a single sequence. In practice, the main benefit of high fragment accuracy is that the score distribution for the random matches drops away. That is, discrimination and sensitivity are greatly improved even though the score for the correct match may have increased only slightly.

Charge

The charge control in the search form set the default charge(s), to be used if a spectrum in the peak list does include charge information. In practice, peak lists almost always include charge information, so the default is never used.

Instrument

The Instrument setting determines the set of ion series that are considered when trying to find a match. Mascot looks for evidence in the spectrum for each series. If no evidence is found, the series is discarded. Many of the instruments are very similar. It only becomes critical to select the correct Instrument is when the data come from an ECD or ETD experiment, which produces c and z ions, not found in most of the other Instrument types. If you are doing alternate CID/ETD, you may need to define an Instrument that is a super-set of both settings. Use the Configuration Editor (link from your local Mascot home page).
 
 
Copyright © 2012 Matrix Science Ltd. All Rights Reserved.