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Mascot searchingalgorithm

Manufactured by Matrix Science

The Mascot searching algorithm is a core component of the Mascot software suite, a leading bioinformatics platform used for the identification and characterization of proteins from mass spectrometry data. The algorithm performs rapid and accurate searches of protein sequence databases to match experimental mass spectrometry data with known protein sequences.

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2 protocols using mascot searchingalgorithm

1

Mass Spectrometry Analysis of Trypsin-Digested Samples

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Following trypsin digestion, samples were freeze dried and resuspended with 25 ul of
5% ACN/0.1% formic acid for mass spectrometry (MS). Peptides were analysed by
LC/MS/MS using a Surveyor LC system and LCQ Deca XP Plus (ThermoScientific). The raw
data files were converted into mascot generic files using the MassMatrix File
Conversion Tool (Version 2.0; http://www.massmatrix.net) for input into the Mascot searching
algorithm (Matrix Science).
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2

Protein Identification via Mascot Search

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The obtained data files (.raw) were converted into mascot generic files using the MassMatrix File Conversion Tool (Version 2.0; http://www.massmatrix.net) for input into the Mascot searching algorithm (Matrix Science). The data files (all merged in one search) were searched against NCBInr (v. 20080527) with plant taxonomy parameter using the following search criteria: tryptic peptides with up to one missed cleavage and carbamidomethylation of cysteines and oxidation of methionines, which were set as variable modifications. In a separate search, individual bands were searched against an in-house database containing sequences of the recombinant proteins. Results were exported to Microsoft Office Excel รข software with a score threshold set at 34; scores above this indicate identity or extensive homology.
The proteins identified in each sample were ranked with the protein with the highest mascot score listed first. The emPAI was also calculated as previously described (Ishihama et al., 2005) . The percentage of the total protein weight contributed by each individual protein was calculated.
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