Label Free Quantification
PEAKS Video Tutorials

Within PEAKS Q is a label free quantification algorithm which uses spectral intensity to determine protein abundance. This is a relative quantification method that finds the quantities of identified peptides based on their intensities in an LC-MS/MS experiment. PEAKS uses the intensity of the precursor ion, more accurately XIC, for quantification.

The following are the six steps involved in the algorithm:

Retention Time Correction: The retention times between the different samples are normalized to correct for differences between LC-MS/MS runs. Dynamic programming is used to maximize the similarities between the MS scans.

Feature Detection: Peptide features (MS peaks that possibly formed peptides) are selected based on isotope distribution and peak shape similarities between adjacent MS scans from different samples.

Feature Vector Formation: Features from different samples with the same normalized retention time and mass are grouped.

Protein Identification: All samples are combined and protein ID is performed on the combined set using PEAKS DB. This has the potential of increasing confidence and coverage. For example, when two peptides of the same protein are identified separately in two different samples, running protein identification in any of the samples alone may result into a low-scoring protein that risks being dropped by the protein identification algorithm. Combining all the datasets together can avoid this pitfall. The excellent scalability of PEAKS Studio is essential for such analysis because the combined dataset is usually large (over 100GB for some of our data). Another important task in this step is the protein clustering. Normally a family of similar proteins can be identified as long as one protein presents in the sample. They need to be clustered together to avoid false positives which complicated the ratio calculation step.

Peptide Ratio Calculation:Peptide ratios are calculated from the feature vectors based on the area under the XIC curve for that peptide.

Protein Ratio Calculation:A weighted sum of the peptide ratios are used to calculate the protein ratio. Normalization is done based on the difference between total intensity of the MS scan from each sample.

What is Required From the Survey Scan for Quantification:

The peptide feature must appear in at least 2 of the samples in the retention time window specified. Also, it must appear in at least three survey scans in the retention time range specified in the run parameters in each sample. The survey scan must be in profile mode, not centroided. There must be at least one protein id result for the peptide.

PEAKS Q - Proteins

Quantified proteins, supporting peptides of each protein and peptide features in the spectrum from each sample are displayed in the result panel. The quantified proteins will appear in the top panel, with homologous proteins clustered together.

PEAKS Q - Peptides

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.

PEAKS Q - Feature Chart

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.

PEAKS Q - Heat Map

Right click your mouse and select “Show features” to display a 2D heat map. The grayscale heat map displays high intensity in black while white represents low intensity. If the peptide is identified in the PEAKS Protein ID, there will be a star after the sample name.

PEAKS Q - 3D View

The “3D View” tab displays 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 and move the cursor to rotate around the image.

Information on Results:

  • Peptide quantification ratios are used to find the protein quantification ratio.
  • The PEAKS Q score is a raw score. The higher the score, the better the result. The PEAKS Q score can only be used to compare different results within a single set of results. It cannot be used to compare two separate quantification runs.
  • The maximum raw intensities of each peptide feature used for quantification can be found in the export. These can be found in the cell adjacent to the peptide ratios.