Label Free quantification relies on the changes in analyte signals directly reflecting their concentrations in one sample relative to another. This technology employs overall spectral intensity normalization by interpreting signals of molecules that do not change concentration from sample to sample. By comparing two or more spectra, PEAKS can determine the constant intensity ratio between the unchanging analytes, the basis for identifying the non-changing concentrations, making spiking unnecessary.

Start by creating a project with two samples. To add the second sample, click the add sample button, and in this case, we’ll declare that both samples came from an FT-trap instrument. Note: For label free quantification to function in PEAKS, you need to have at least 1 sample with at least 1 file/fraction in each sample. Now, let’s use the workflow feature used in the previous quantification tutorials.

Click on the workflow button and select Quantification.

Click “Select Data” and find our label free project.

Click “All Samples” and then “Add to Right”. The samples should now appear in the “Selected Data” panel as shown. 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 parameters:

• Correct precursor charge states: 1 to 4
• Filter Scans: quality value greater than 0.65
• Preprocess the data: Yes

Click “OK”.

Upon review, click “OK”.

Check the 4th box and then click the “PEAKS Search” button. Call up the label free parameters and set our database to Sample DB.

Check the 5th box and then click the “Quantification” button. Select “Label Free” from the left hand panel and enter the following parameters:

• Mass error tolerance 0.2 daltons
• 4.0 minutes
• Peptide score between 0.5 and 1.0
• Protein score 0.5 – 1.0.

Once the workflow analysis is complete, you should see the following in your “Project View” panel.

Double click on the label free node. The identified proteins will appear in the top panel, with homologous proteins clustered together. The ratio of Sample 1: Sample 2 appears in the “Ratio” column and the standard deviation of Sample 1: Sample 2 appears in the “SD” column.

In the example below the standard deviation is 0 as there is only one supporting peptide for the protein shown.

The supporting peptide is shown under the “Peptides” tab. The retention time is shown for the specific peptide as well as the peptide ratio from Sample 1: Sample 2.

Click on the “+” beside the “Outlier” folder to see the peptides that were not included in the ratio.

To see which peptides were used to identify the protein during the PEAKS protein ID search, select the “Coverage” tab. The entire sequence of the protein is shown and the matching peptides are highlighted in blue. In this example the total matched part accounts for 9.392% of the protein. This information can be found in the “Coverage” column in the “Protein View” panel.

The features chart will appear by default in the bottom panel.

To see exactly where the selected supporting peptide corresponds to the protein sequence, select the “Peptide detail” tab. The entire sequence of the protein is shown and the selected supporting peptide is highlighted in blue.

Clicking on the “Quantification” tab will display a 2D view of the features. Move your cursor around the map to display the m/z ratio and retention time.

Right click your mouse and select “Show features” to display a 2D heat map. When viewing the heat map in color, red represents high intensity and yellow represents low intensity. The grayscale heat map displays high intensity in black while white represents low intensity.

Click on the “3D View” tab to display a 3D View of the peptide features for sample 1 and sample 2. Intensity is displayed on the y-axis, m/z on the x-axis and retention time on the z-axis. Click on the image and move the cursor to rotate features.