Peptide mass fingerprint search

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-stain-ing, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method. 

Keywords Proteomics MALDI-TOF mass spectrometry SDS-PAGE 2D-gel in-gel digestion peptide mass fingerprint protein identification database searching. 

Fig. 3. Mascot peptide mass fingerprint (PMF) search page.

Fig. 4. Mascot search results summary page using peptide mass fingerprint (PMF) data from the spectrum in Fig. 1.

An important development in high-throughput protein identifieation is the introduction of protein database searching [111]. After separation on ID- or 2D-GE, the proteins were blotted onto a membrane and enzymatically digested after reduction and alkylation. The tryptic peptide mixture is analysed by MALDl-MS to achieve a peptide map or peptide mass fingerprint (PMF). The m/z information of the peptides is used to search the protein database, e.g., the Protein Identification Resource (PIR) database [112-114]. If the mass of just 4-6 tryptic peptides is accurately measured (between 0.1 and 0.01%), a useful database search can be performed. 

Database searching with m/z data from peptide mass fingerprints (PMF) or uninterpreted MS-MS spectra was pioneered by several groups [4-6]. 

Several software search engines, including MS-Fit, MOWSE, Prot-ID, Expasy tools, ProFound, Mascot, and PeptideSearch, are available for use with the peptide-mass fingerprinting data. During the search process, the algorithm matches the measured molecular masses of peptides in the query against the list of theoretical mass... 

MOWSE (http //www.seqnet.dl.ac.uk/Bioinformatics/Webapp/mowse/) at the SERC Daresbury Laboratory, a peptide mass fingerprinting tool searching the OWL database. 

Mascot (http //www.matrixscience.com/cgi/index.pl page=/search form select.html) at Matrix Science Ltd. which provides identification resources for peptide mass fingerprinting, MS/MS ion search and MS/MS sequence queries. 

Figure 3. Identification of a protein by peptide mass fingerprinting. The protein constituents of pig saiiva were separated by SD-PAGE and a protein band was digested with trypsin. The resuitant tryptic peptides were mass-measured using MALDI-ToF mass spectrometry. The peptides in the mass spectrum were either derived from trypsin self-digestion (T) or were derived from the protein in the gel- Database searching with the masses of these peptides led to an unequivocal identification of the protein as SAL (salivary lipocalin). The inset map shows the theoretical tryptic digestion map of this protein, and underneath are the peptides that were observed. In many instances, smaller peptides were visible as partial digestion products.

---