Mascot search

Protein Identification Using MALDIPMF tsnJxccJ Mascot Search Results... 

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

Fig. 5. Detailed information for the protein gi 4389275 identified in the Mascot search results in Fig. 4.

The power of the currently available methods for proteome mining can be illustrated with data reported on the identification of 800 proteins from 50 milhon E. coli K12 cells using multiple fractionation, RPLC-MS-MS, and various ways of database searching [37]. With MASCOT searching, 754 proteins were identified from 1326 peptides and 2167 MS-MS spectra, which means on average 1.75 peptides and 2.87 spectra per protein. Abont 17% of the E. coli K12 proteome was covered in this way. 

In another study, related to the E. coli K12 proteome, by another group [37], the tryptic digest was first fractionated in 40 fractions by RPLC. Next, Met-containing peptides in these fractions were oxidized by HjOj. These modified proteins were separated from the bulk unmodified proteins by RPLC and fractionated in 8 fractions. These fractions were subsequently analysed by nano-LC-MS-MS on a Q-TOF instrument operated under DDA. After the first RPLC run, an exclusion hst was generated from peptides identified in a MASCOT search of the data, and the sample was then remn. Following this procedure, 754 proteins were identified, which is about 34% of the expressed E. coli K12 proteome. 

Fig. 1. Identification of m/z 1465 from proteomic profile. Panel A shows the TOF/TOF spectrum of m/z 1465 annotated in BioTools (Bruker Daltonics, Inc.). Panel B shows results from the MASCOT search engine.

Database search parameters for the TOF/TOF data using the Mascot search engine Homo sapiens for taxonomy, NCBIr for database, no enzyme, no modifications, mass tolerance of the precursor ion at 0.2 kDa and the fragment ions at ... 

Figure 3.10 Protein identification by MALDI-MS peptide mass mapping. The MALDI-MS peptide mass map was used to search a database using the Mascot search engine. The highest scoring protein sequence (here a mouse protein) allows the...

Figure 10.3 An example of peptide identification with MS/MS. For a peptide of interest at m/z 1 777.97, an MS/MS spectrum is acquired at the precursor mass using the biological sample with the highest intensity level for the peptide. The MS/MS spectrum is searched against a database of human protein sequences using the MASCOT search...

Recently, Tracz et al. (2013) reported a novel variation of the above method for bacteria identification implemented by using the Mascot search engine. In this approach, they compared the nmnber of peptides shared between the unknown and DB strains by tricking Mascot to report such assignments. They achieved it by creating pseudo-polyproteins of concatenated protein sequences of each DB strain proteome (see Section Bacterial DBs for details), so searches against such a DB report PSMs to DB strains instead to particular proteins. 

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