SEQUEST algorithm

Several algorithms are available for the analysis of MS/MS spectra including SEQUEST, MASCOT, and X Tandem among others. Note that additional secondary quality control of assessment of MS/MS data has recently been implemented to assess identification probabilities and false positivity rates. The MS/MS spectra from an experiment can be interrogated against a concatenated forward and reverse database and an assessment of the intrinsic error rate of the data set can be made. Other approaches for secondary analysis of matching scores for peptide sequencing data include XCorr score normalization routines that are independent of peptide and database size.33... 

A complementary approach to protein identification is based on peptide seqnencing analysis (PSA) by collision-induced dissociation (CID) in MS-MS. Por proteomics studies, the de novo sequencing of MS-MS spectra is too time-consuming. Therefore, algorithms have been developed to either provide antomated MS-MS spectmm interpretation, or perform protein identification by means of a SEQUEST database search [6-7]. In the latter case, the precursor m/z values are matched against a virtual digestion of all the proteins in the database. Sequence ions are predicted for the peptides that match the precnrsor m/z values, and compared with the measured MS-MS data. A correlation score is calculated for each match. 

The SEQUEST database search algorithm (Ch. 17.6.2) is a powerful tool in protein identification, although like any statistical tool it relies on the jndgments of the user to critically evaluate its results. Problems may especially arise, when unanticipated modifications are present in the protein investigated. 

In the case of the ESTMS-MS data, actual amino acid sequence can be deduced. This is possible due to the CID processes, which breaks the peptides further into amino acid ions. Each amino acid ion has a specific mass and by calculating masses of specific amino acid from the MS spectra, the exact sequence of the peptide and in turn the protein can be deduced. The workhorse of such analysis is a program called Sequest . Since the ESTMS-MS analysis provides information about the actual amino acid sequence, it is also useful to obtain information about protein modifications (such as phosphorylation) and toxicant-induced protein adducts. This has become even easier with the advent of new software tools and highly intelligent algorithms such as SALSA . 

A method developed by Eng and Yates uses a MS/MS cross-correlation algorithm called SEQUEST . - In this method, an unknown protein is digested using trypsin and the resulting peptides are used to collect product MS-MS scans. These MS-MS scans are used to search a protein database for a match within the expected MS-MS data from the known tryptic peptides. 

Candidate peptide sequences from the mixture are later identified using a computerized database search algorithm (e.g., SEQUEST) [14] and validated using the STATQUEST probabilistic scoring program [15]. 

MacCoss, M.J. Wu, C.C. Yates, J.R. Probability-based validation of protein identifications using a modified SEQUEST algorithm. Anal. Chem. 2002, 74, 5593-5599. 

The MS instrument returns LTQ or LTQ-FT raw data files that contain the mass spectral data. These may be analyzed by Bioworks, that features the SEQUEST [20] protein search algorithm for protein/peptide identification. The predicted peptides in the search analysis are verified for the fragmentation pattern in the MS/MS spectra. Alternatively, the data can also be uploaded to the Mascot server [21], and analyzed for peptides and corresponding protein identification. 

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