As the first steps are loading a database and creating a project, which were addressed in the first video, just now with a different sample file, SILACsample.mzxml found in our PEAKS program folder, we are going to start this video with the SILAC file already loaded up into PEAKS. Let’s use the workflow feature to save us some time.

Click on the workflow button and select Quantification

Click “Select Data”; select the SILACSample.mzXML file and then click “Add to Right”.
The samples should now appear in the “Selected Data” panel.

Click “OK”.

The workflow window should now display “finished” beside the “Selected Data” button.

Check the 2nd box and then click the “Refine data” button.

Enter the following parameters:

• Correct precursor charge states from 1 to 3
• Preprocess data, yes

Check the 3rd box and then click the “de novo” button.

Enter the following parameters:

• Parent ion 0.1
• Fragment ion 0.8
• Enzyme is trypsin
• Oxidation M and K6 as variable modifications
• Carbamidomethylation as fixed

Save these parameters as SILAC.

Click “OK”.

Check the 4th box and then click the “PEAKS Search” button. Load the SILAC parameters and make sure our sample db is our database, click OK.

Check the 5th box and then click the “Quantification” button. Select “Label at the MS level” from the left hand panel and enter 0.1 as mass error tolerance, upper bound of precursor charge as 4, retention time as 1.0 and peptide score threshold as 0.6. Add our labels by clicking "Add label" and identifying the first one as light with a mass of 0.0 and the second as heavy with a mass of 6.0.

Click “OK” and then click “Start” in the workflow configuration panel.

Click “Start Jobs”.

Once completed, the protein quantification result will be displayed in the PEAKS Protein ID node.
Double click on this node the “Peptide View” results will appear by default.

Click on the “Peptide Details” tab to see where the selected peptide matches the protein highlighted in blue.

PEAKS provides the 3D View of each peptide feature for visual validation. Select the “3D View” tab. The panel along the bottom allows you to narrow in on the peptides that you would like to examine. You can specify a particular scan number range, m/z range or intensity range. Click the “Apply” button to change the 3D View to your specified values.

Select the “Protein View” tab.
The SILAC quantification results are listed as a “Ratio Heavy: Light” and “Standard Deviation Heavy: Light”.

Note that the top protein result is Human Filamin A, with a score of 98.89%. The ratio of Heavy: Light is highlighted for each protein in the red box below. For example, the highest ranked protein, Human Filamin-A has a ratio of 1.2 and a standard deviation of 0.13.

A ratio of -1 means that while a reporter ion was found for the selected peptide, a ratio could not be calculated for the protein as the peptide did not meet the required score criteria (in this case we specified that the peptide score had to be greater than 0.6). A blank box in the ratio column means that a reporter ion for the selected peptide was not found.

There are four peptide features are identified and calculated for quantification of Human Filamin-A. Two other peptides (Spectrum 40 and 49) were used to identify the protein but were not identified as features.

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