SEQUEST is the original, and still one of the leading, programs that correlate uninterpreted MS/MS spectra of peptides with the amino acid sequences from protein databases. If you want to understand what SEQUEST is telling you, look at the interactive Sequest Browser summary page. SEQUEST is the search engine sold by Thermo Finnigan with its mass spectrometers. Sorcerer, a special hardware platform for running an optimized version of SEQUEST, is available from Sage N Research. DtaSelect is a program that organizes Sequest results. The Wildcat Toolbox from the Arizona Proteomics Consortium has various perl programs for further organizing Sequest, X! Tandem and FASTA files.
Mascot is a widely used program that also uses mass spectrometry data to identify proteins from protein databases. Mascot has a extensive set of web help pages that includes a lot of information on protein identification by MS. You can use Mascot, in a limited way, on the web for free.
X!Tandem is a very fast seach engine which searches for matches of MS/MS spectra to a protein database. X!Tandem is free to use on the web or to download. X!Tandem works in conjunction with thegpm.org and can take advantage of the information gleaned from previous searches. It has an option, PPP, which searches first only the peptides that have been found most frequently for each protein. This can speed searches up by a factor of 20.
OMSSA is another free search engine for identifying MS/MS peptide spectra. OMSSA was developed by the NIH and is completely free. You can find both X! Tandem and OMSSA as well as other proteomics software at the ProteomeCommons web site. Another search enigine is Silver. Silver is a program at Yale that uses peak intensities as well as m/z information..
Scaffold enchances the results of a SEQUEST or Mascot search using Bayesian statistics and X!Tandem to increase the number of reliable identifications and decrease the number of false identifications.
Phenyx is the newest of the major commercial search engines. It boasts a family of scoring functions that you can tailor to your specific needs. Phenyx has a nice user interface which is well integrated with Swiss-Prot. Phenyx also has a on-line version that you can try for free on the web.
Spectrum Mill is the peptide search engine that Agilent sells with its mass spectrometers. Spectrum Mill implements a workflow for the MS experiment. First Spectrum Mill does an assessment of the quality of the spectra and only searches the worthwhile spectra. It also validates the spectra both automatically and visually.
ProteinLynx is the peptide search engine that Waters Micromass sells with its mass spectrometers. ProteinLynx manages a workflow which passes spectra not identified in the first search to a second or third search. For example the first search might be unmodified proteins, the second proteins with modifications and the third de novo sequencing followed by BLAST.
InsPecT is a program that partially interprets an MS/MS spectrum to derive short tags and then searches a protein database with these tags. GutenTag is a similar program. If you have the tags, then MS-Tag, PeptideSearch, or ComboSearch will use the tags to search a database.
Peaks takes MS/MS spectra files and does de novo sequencing to directly figure out what the amino acid sequence of the peptide is. Lutefisk and PepNovo are well regarded de novo sequencing programs that are available for free. De novo sequencing is also a part of Spectrum Mill and ProteinLynx and is also available from Thermo as denovoX.
Spider is a program to search protein databases for the sequences produced by de novo sequencing programs such as Peaks, Lutefisk or PepNovo. For more information see the What is Spider? page.
If you have a peptide's sequence of amino acids, and want to see what the y and b ions are, use the Fragment Ion Calculator or Ion Predictor. This is useful if you want to understand the peaks that are labeled by SEQUEST or one of the other search engines.
The standard MS/MS experiment starts by digesting the proteins into peptides with an enzyme such as trypsin. To see what peptides will result from digesting a protein, use the Protein Digestor or the equivalent here. You can download a similar program, Paws.
In top down proteomics, an alternative way of gaining insight into a proteome, whole proteins are run through a mass spectrometer. ProSightPTM is the only software available for top down protein identification and characterization.
A proteomics experiment involves much more than just mass spectrometry. There are also numerous steps of sample preparation, enrichment and/or depletion, separation and/or pooling. In additon as you can see above, there are a number of software processing steps. Laboratory information management systems (LIMS) can track all these operations. Proteus from Genologics is a LIMS oriented toward proteomics and mass spectrometry.
The retention time of the reverse phase liquid chromatography commonly used with electrospray ionization depends upon the hydrophobicity of the peptide. You can use this profiler to verify that the peptides identified are eluting in a reasonable order. You may want to download Winpep or buy GPMAW to examine the characteristics of your peptides.
This tool computes the theoretical pI (isoelectric point) and Mw (molecular weight) for a list of Swiss-Prot proteins or for a sequence of amino acids. You can use this to check that the identified protein is consistent with its position on a 2D PAGE gel. Remember that real proteins in samples are often modified in ways that change pI and Mw from these theoretical values.
