MS/MS data obtained using nano-LC-MS/MS is searched against the predicted fragment ions from the trypsin digestion of proteins contained in a database such as NCBInr using Mascot. The application of certain constraints such as mass accuracy limits of the instrument, narrowing down taxonomic category, specifying modifications on residues etc. help make the search more efficient. Most successful identifications are generally made from the fragmentation spectra of 2+ and 3+ ions of peptides generated by electrospray ionization. There are many spectra that do not result in a successful identification due to the poor quality of the fragmentation pattern achieved. Sometimes a poor fragmentation is due to the charge state of the ion (>3+ or 1+), specific sequence of an ion or simply poor sensitivity. However, since a protein can be identified based on the detection of as few as 2 peptides, most samples submitted in sufficient quantity provide the identification of at least one protein. Since each ion is isolated first and then fragmented, an individual peptide fragmentation pattern provides information independently about its protein of origin. This means that multiple proteins from a mixture can easily be identified during the course of a run. However, as the complexity of the sample increases, there is simply not enough time to perform MS/MS on all the peptides and therefore, the number of proteins that can be identified from a single reverse phase LC run is limited.

     A detailed report in Adobe Acrobat (.pdf) format is provided to the users through the “paperless” system. The first page (Figure 1) contains a summary of search parameters used and a link to the Mascot results file. This file is a multilevel/interactive report that contains extensive information including probability based Mowse scores for all proteins identified with >95% confidence level, MS/MS spectra, sequence coverage, ion scores for top few peptide sequences for each identified peptide, and direct links to supplemental information provided in the databases etc. Results can be viewed in different formats and with variable confidence levels. For a detailed description of the Mascot results file please see Results Interpretation.