The following procedure should be followed to calculate the relative ratios of phosphopeptides between isotopically-labeled samples (i.e. SILAC or 15N). For phosphopeptides with only one phosphorylated residue, this corresponds to quantitation of the phosphorylation site. Click “Run now” in the “Quantitative Analysis” column for the associated search and filtering results.
Figure 9.6.1: Run now lin in quantitative analysis column
Select the appropriate labeling method. For this example we used 15N. For a description of procedures for other labeling methods see Section 8 of this manual.
Figure 9.6.2: Labeling method selection
The default quantitation values for high mass accuracy data (e.g. Orbitrap) and appropriate masses differences for each amino acid will appear in the following table. Click “Submit”.
Figure 9.6.3: Quantification parameters
After the Census quantitation from MS1 chromatograms is complete, the following links will be available for both viewing quantitative results for the run (Quantitative Analysis – View data) and validating individual peptide quantitation values (Census – Launch Census).
Figure 9.6.4: View data and launch census links
Quantitative comparison and filtering of statistically confident phosphopeptides is done as follows:
The quantitation©COMPARE tool can be used to quantitatively compare phosphopeptides, filter statistically confident phosphopeptides, and perform label-free comparison by spectral counts. To run, click on the “quantitation©COMPARE” link.
Figure 9.6.5: Quantitation ©COMPARE link
The following screen appears with a link to run a new comparison. Click on “run new quantitative ©COMPARE.”
Figure 9.6.6: Run new quantitative ©COMPARE link
The list of experiments which have had PhosphoAnalysis run on them within that project will be listed with relevant experimental information. Start by entering a name for the replicate experiments to be combined for statistical quantitative comparison in the “group name” entry box. Next, select the experiment to be combined by checking the boxes next to the experiments as shown below. Then click “Add to group.” The next screen that appears will have the first “Group name” listed of experiments that were combined on the previous page. Repeat the selection of experiments to be grouped for quantitative comparison to another group. Click “Add to group” again and repeat this process until all of the comparisons to be performed are grouped. Then click “Next”.
Figure 9.6.7: Add to group selection
The following screen will appear. Name the quantitative comparison in the “Give a name to save” entry box and click “Run it now.”
Figure 9.6.8: Saving quantitative comparison names
The following screen appears with the previously performed comparisons:
Figure 9.6.9: View quant COMPARE page
Click on the “ANOVA comparison” link to view the quantitative comparison results with the following format:
Figure 9.6.10: ANOVA comparison results
This page lists:
- The uniqueness of the phosphopeptide (Unique column).
- The most confident phosphorylation site (Corrected phosphopeptide column).
- Sequences before localization analysis.
- The localization confidence (Localization Score) – if greater than 13 there is 95% confidence in the phosphorylation site and if greater than 19 there is 99% confidence in the phosphorylation site.
- The phosphopeptide confidence (Debunker Score) – the value directly corresponds to the confidence on a scale of 1 for phosphoserine and phosphothreonine.
- The files which contained the spectra for the identified phosphopeptide (Filename).
- The conventional peptide identification scores (XCorr, DeltCN, Conf%, M+H+, CalM+H+, TotalIntensity, SpR, Prob, IonProportion).
- The spectral counts of the phosphopeptide in different analyses (Spec Count).
- The protein origin of the phosphopeptide (Proteins and Protein Descriptions).
A key feature to this page is the ability to actively filter the phosphopeptides quantified for comparison based on their Localization Score, Debunker Score, or both. To do this, enter the desired cutoff value for either or both in the entry boxes and click “Filter.” If the Ascore is greater than 13 there is 95% confidence in the phosphorylation site and if it is greater than 19 there is 99% confidence in the phosphorylation site. While the phosphopeptide confidence value (Debunker Score) directly corresponds to the confidence on a scale of 1 for phosphoserine and phosphothreonine
Figure 9.6.11: Ascore and Debunker score filter
After filtering as shown above, the results can be exported by selecting one of the options at the bottom of the page as shown below: