Validation of MS/MS Data

Why do you need to validate search results? 

data validation chartThe results of a database search are usually characterized by individual peptide/protein scores. The scores help to discriminate between correct and incorrect peptide assignments to spectra and therefore facilitate detection of false protein identifications. However, there are no universal criteria based on the database search scores for sorting out correct identifications from incorrect ones. Besides, while most of the database search engines apply similar methods to assign peptides from a sequence database to experimental MS/MS spectra, they all have different algorithms for calculating peptide scores. These scores are not equivalent and can not be compared directly. 

To improve peptide identification accuracy, coverage and confidence the APL combines the results of protein identification by multiple search engines (Mascot, Sequest and X!Tandem) using Scaffold software. Scaffold computes probabilities that peptide assignments to spectra are correct based upon database search scores and the number of tryptic termini of peptides. The results of such statistical analysis can be compared between different research groups, using different mass spectrometers, and different database search software.


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