CRP

CRP predicts protein phosphorylation sites by simulating in silico proteolytic cleavage and Edman sequencing outcomes for 32P-labeled phosphoproteins to support identification of phosphorylated Ser, Thr, or Tyr residues.

Key Features:

• In Silico Proteolytic Cleavage: Performs computational proteolysis on input protein sequences to predict peptide fragments that would result from specified proteolytic reagents.
• Edman Cycle Radioactivity Prediction: Predicts which Edman sequencing cycles would release 32P and thus exhibit radioactivity for candidate phosphorylation sites.
• Comparison with Experimental Data: Compares predicted Edman cycles containing 32P with observed experimental cycles to confirm candidate phosphorylation sites and resolve ambiguities.
• Residue Specificity Filtering: Allows specification of phosphorylated residue type (P-Ser, P-Thr, or P-Tyr) to exclude alternative sites inconsistent with experimental conditions.
• Kinase Recognition Motifs: Incorporates known kinase recognition motifs into predictions to provide additional evidence for likely phosphorylation sites.
• Experimental Design Assistance: Suggests combinations of proteolytic reagents for additional cleavage experiments to discriminate among multiple candidate sites.
• High Sensitivity Complement to Mass Spectrometry: Provides a high-sensitivity approach that complements mass spectrometry and can operate at femtomole protein levels and minimal radioactivity (e.g., ~1000 counts per minute).
• Database Coverage Assessment: Analysis against the PhosphoBase sequence database indicates unambiguous identification of ~60% of known sites from two cleavage experiments, rising to ~70% with additional experiments.
• Routine Differential Proteolysis Application: Supports routine determination of in vivo phosphorylation sites through differential proteolysis analysis and Edman cycling data interpretation.

Scientific Applications:

• Phosphorylation Site Identification: Identifies candidate phosphorylated Ser/Thr/Tyr residues from sequence data combined with Edman cycle radioactivity measurements.
• Ambiguity Resolution: Resolves ambiguous phosphorylation-site assignments when multiple residues could account for observed 32P release.
• Experimental Planning: Guides selection of proteolytic reagents and experiment combinations to discriminate among candidate sites.
• Complement to Mass Spectrometry: Provides corroborative high-sensitivity evidence for phosphorylation sites detected or missed by mass spectrometry.
• Database-scale Assessment: Enables evaluation of expected site-identification coverage across protein sequence databases such as PhosphoBase.

Methodology:

Performs computational proteolysis on protein sequences, predicts Edman sequencing cycles that would release 32P for candidate phosphorylated Ser/Thr/Tyr residues, compares predicted cycles to experimental Edman-cycle radioactivity, allows residue-type filtering, incorporates kinase recognition motifs into predictions, and suggests combinations of proteolytic reagents for further experiments.