9.3 Validation of Phosphopeptides and Phosphorylation Sites

DTASelect is used to filter modified and unmodified peptide sequences from IP2 search results. Ascore can be used for phosphorylation site localization. After selecting the DTASelect filter, run “PhosphoAnalysis” by clicking “Run” as shown below:

Figure 9.4.1: Experiment screen with Phospho-Analysis “Run” link

The following screen should appear where “Run” should be clicked again: (Note: For phosphor-peptide quantitation, PhosphoAnalysis MUST be run before Quantitative Analysis.)

Figure 9.4.2: Run Phospho-peptide Analysis

When the analysis is complete the “View” and “Rerun” links will appear in the “phosphoanalysis” column:

Figure 9.4.3: View and Rerun links

Upon clicking “View” in the “phosphoanalysis” column the following screen will appear:

Figure 9.4.4: Phospho-analysis Search Result

This results screen lists the following information:

• 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 DTASelect confidence score.
• 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).

Click the phospho (protein) button to display PTM results in protein level.

Figure 9.4.5: Phospho-analysis Search Result

This results screen lists the following information:

• Protein accessions
• PTM sites based on protein sequence
• PTM site index, modified spectral count, unmodified spectral count, average localization score of multiple spectra
• Site centered sequences