Analyzing Protein Identification Results

Protein identification/quantification results are stored in a relational database and the results are available for viewing and analysis via web access. The database and viewer are part of the Computational Proteomics Analysis System, or CPAS. Access to CPAS can be found at:

https://proteomics.fhcrc.org/CPAS/Project/home/home.view

In the right hand corner of the webpage is a link called "login". The login page will require a username and password. If you do not already have access to CPAS, contact the Proteomics Facility to set up an account for you.

Using the CPAS Viewer

After logging in to the CPAS site, your username should be visible on the left-hand side of the page. Click on your username and the general peptide summary page will be visible. This page shows all of the tandem mass spectra that were subjected to identification by database comparison. As is typical with most LC MS/MS techniques, a good portion of this data is "bad" while a minor portion is "good". The trick is to filter the peptide results to remove most of the bad identifications while keeping most of the good identifications.

The peptide filtering criteria described below are suggested based on experiences from the Proteomics Facility. These criteria should be considered as guidelines for filtering peptide identification data. Users may wish to change the stringency of the filtering criteria based on their experience and need.

Standard Peptide Filtering Parameters

1. Peptide Prophet- In the PepProphet column, click the triangle and select "Is greater than" from the pull down menu. Then type in 0.9 in the value box. Click okay. Peptide identifications with Peptide Prophet scores less than 0.9 will be removed.
2. Ion%- Click the triangle next to "Ion%" in the Ion% column. In the dropdown box, select "Is greater than or equal to" and add "0.30" to the value box. Select "okay" and all peptide identifications that have %ion values less than 30% will be removed.

It is important to note that a summary of the filtering criteria applied is summarized on the line labeled "Peptide Filter".

Grouping Peptides into Proteins and Filtering Proteins
Clicking the text "Collapsed Protein View" or "Expanded Protein View" provides two views after grouping the filtered peptides into proteins. Additional filtering can be performed to remove those proteins that are identified by a single peptide. This is done by selecting the triangle in the "unique" column. In the first drop down box, select "Is greater than" and add "1" to the value box. Select "okay" and all proteins that are identified by one peptide will be removed.

Saving view
All filtering and viewing parameters up to this point can be saved. In the row called "view", select "save view". Type in a name and click "save". This view can then be applied to any file at any time to help minimize key strokes and to ensure all result files are treated identically.

Protein Summary
Each protein name in blue can be clicked to gain additional information about the protein and the identification results. Within the summary, the peptides that were identified in the tandem mass spectrometry experiment are listed. The scan number of each peptide can be clicked to view a comparison of the tandem mass spectrum of that peptide to the expected fragmentation mass fragments of that peptide.

Note: This description of how to use CPAS is meant to only get new users familiar with the viewer and to get initial analysis of the results started. CPAS has many tools built into the viewer that can be helpful with analyzing the results. Help files are available in CPAS and Proteomics Facility staff is available to help with using CPAS.

	Analysis of Results