As the first steps are loading a database and creating a project, which were addressed in earlier tutorials, just now with a different sample file, itraqsample.mzxml found in our PEAKS Program Folder, we are going to start with this file already loaded up into PEAKS. Let’s take this opportunity to touch upon the workflow feature of PEAKS.

Click on the workflow icon and select Quantification.

Click “Select Data”.

Select the iTRAQSample.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.

Correct precursor charge states from 1 to 3, and preprocess the data. Click “OK”. Check the 3rd box and then click the “de novo” button. Enter the parent ion error tolerance to 0.3 daltons, and fragment ion to 0.1 daltons, enzyme to trypsin, and then our PTMS. Applied Biosystems ITRAQ 4plex Y as variable, Applied Biosystems ITRAQ 4plex N as fixed, Applied Biosystems ITRAQ 4plex K as fixed, oxidation M as variable and carbamidomethylation as fixed. To save these parameters, click the save as button, give the parameters a name, like ITRAQ, and go back into the workflow configuration window and click “OK”. Check the 4th box and then click the “PEAKS Search” button. To save us time here, lets pull up the predefined ITRAQ parameters, then set our database to sample db and click OK.

Check the 5th box and then click the “Quantification” button. Select “Label at the MS/MS level” from the left hand panel and enter the following parameters, basic options, mass error tolerance to 0.1 daltons, upper bound of precursor charge to 4, peptide score threshold to 0.6. Down at the bottom click add label, call it S1 for 114.112 and S2 for our reporter ion 117.115.

Click “OK” and then click “Start” in the workflow configuration panel. The following window will open. Click “Start Jobs”.

Once completed, the protein quantification result will be displayed in the PEAKS Protein ID node . Double click on this node and the “Peptide View” tab will appear by default. The quantification results are listed as a “Ratio of 117.115:114.112”.

Click on the “Peptide Details” tab to see a simple alignment between the original de novo sequence, the peptide found in the database and the reconstructed sequence. At the bottom of the “Peptide Details” panel you will see where the selected peptide matches the protein highlighted in blue.

PEAKS provides the 3D View of each peptide feature for visual validation. Click on 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 quantification results are listed as a “Ratio of 117.115:114.112” and as “Standard Deviation of 117.115:114.112”. They are highlighted in the red box below. For example the relative protein ratio for the top ranked protein (Beta-galactosidase) is 3.01 with a standard deviation of 0.45.

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