Further Analysis of Mass-Spectrometry data

The next stage is to analyze the peptides and proteins identified by the search engines. For this tutorial, we will look in detail at the peptide summary report produced by Mascot. We will also look at how modifying the search parameters impacts the results.

Exploring Mascot results

1. Explore the various components of the peptide summary.
2. Try some of the following modifications:
   • Select fixed or variable modifications (e.g, try phospho ST or phospho Y).
   • Select the Error tolerant checkbox.
   • Select the decoy checkbox to search "decoy" databases.
3. Explore the impacts of these modifications on the results. Are there any hits?

Exporting protein lists from a search engine

In the Mascot Peptide Summary Report:

1. In the Format As dropdown list, select Export Search Results.
2. Click Format As.
3. In the Export Format dropdown list, select CSV.
4. Ensure that the Protein Hit Information checkbox is selected, and that all other sections are deselected.
5. Click Export Search Results.
6. Save file to appropriate location.

In the X!Tandem results page:

1. in the display as: drop down menu, select excel

Note:

The results may need to be reformatted (gene list extracted) or identifiers mapped to gene symbols or entrez gene IDs for use in some of the biological analysis tools.