Phosphopeptidecontaining

Phosphopeptide-containing peptides are peptides that carry a phosphate group on an amino acid residue, most commonly serine, threonine, or tyrosine. Phosphorylation is a common post-translational modification that regulates protein activity, interactions, localization, and turnover. Because phosphorylated sites are often substoichiometric and dynamic, detecting and mapping phosphopeptides is a key focus of phosphoproteomics.

In proteomic workflows, phosphopeptides are enriched before mass spectrometry to overcome low abundance. Common enrichment methods include immobilized metal affinity chromatography (IMAC), titanium dioxide (TiO2), and polymer-based approaches. Fragmentation techniques such as collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), or electron-transfer dissociation (ETD) help localize the exact phosphosite within a peptide.

Data analysis involves site localization scoring, such as Ascore or ptmRS, to assign confidence to the phosphorylation site. Databases like PhosphoSitePlus and Phospho.ELM curate experimentally observed phosphosites. Researchers use phosphopeptide data to reconstruct signaling pathways, study responses to stimuli, and investigate disease-associated phosphorylation changes.

Technical challenges include labile phosphate groups that may be lost during fragmentation or sample handling and the need for accurate quantification of phosphorylation occupancy. Ongoing improvements in instrumentation, enrichment chemistries, and computational tools continue to expand the depth and reliability of phosphopeptide identification.