Tutorial - SPIDER

This video is going to address the SPIDER tool, used for sequence reconstruction. In this example, spectrum 6 has not been identified with database searching tools. In order to gain more information from our data, let’s run a SPIDER search.

1) Click on the Protein ID result node
2) Next, click the SPIDER Search toolbar icon or select “SPIDER Search” from the “Tools” menu.
3) Enter the settings:

  • Homology Match
  • Report top 5
  • Mass tolerance 0.1 daltons
  • Deamidation and Oxidation M as variable modification
  • Carboxymethyl as fixed
Parameters can be saved for future reference by clicking on the “Save Parameter” button.

After the SPIDER search has completed, double click on the SPIDER results node.

The “Peptide View” window will appear. The format is identical to what was seen in the results of a Protein ID search.
Note that all spectra can now be identified by the SPIDER search. Notice how spectrum 6 is well identified.

Clicking on the “Peptide Details” tab will display the protein with its matched peptides in red. SPIDER will also display a reconstructed sequence.

Click on the “Protein View” tab and then on the “Coverage” tab to see where the SPIDER results overlap with the protein sequence. Note that SPIDER matches are shown in red while matches found by both PEAKS protein ID and SPIDER are shown in magenta.

Click here to continue on to the next tutorial, dedicated to iTRAQ Quantification.

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