Comparison of phosphopeptides is accomplished through the identification©Compare tool, as shown below:
Figure 9.5.1: identification©Compare link
The identification©Compare tool can be used to assess the overlap of phosphopeptides between analyses, filter high confidence phosphopeptides for comparison, and perform the label-free comparison by spectral counts. After clicking on the “identification©Compare” link, the following screen appears with a link to run a new comparison and lists the previously performed comparisons. Click “run new identification ©COMPARE:”
Figure 9.5.2: run new identification©Compare
The following screen allows for the selection of different phosphopeptide experiments to compare. To perform a phosphopeptide comparison, select “phosphopeptides,” then select the checkboxes for the experiments to be compared, and click “Run.”
Figure 9.5.3: Selecting phosphopeptide comparison
After clicking “run” in the final step above, IP2 will return you to the comparison page where the results will be listed as shown below:
Figure 9.5.4: Phosphopeptide comparison results
The results can be viewed either as a Venn diagram or as results page by clicking on “Venn Diagram” or “view”, respectively. A new page will open to display the Venn diagram. The text and circles can be rearranged by left or right-clicking and holding down while moving. The image can be printed to a PDF or a printer by clicking “Print.”
Figure 9.5.5: Phosphopeptide comparison Venn diagram
The view page should be similar to the below figure, and lists the following items:
- The uniqueness of the phosphopeptide (Unique column).
- The highest confidence phosphorylation site (Corrected phosphopeptide column)
- The original identified phosphorylation site (Original phosphopeptide column).
- 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).
Figure 9.5.6: Phosphopeptide comparison “view results” output
The web-based results can be sorted by any column by clicking on the column title. These results can be exported to view in other programs as comma-separated-values, Microsoft Excel, XML, or Adobe PDF files using the different “Export options” at the bottom of the page:
Figure 9.5.7: Export options
Alternatively, these results can be copied from within the window that opens when the “view peptide count COMPARE file” link is clicked. The link is shown in the diagram below, and output results in the following figure:
Figure 9.5.8: View peptide count COMPARE file link