Phosphopeptide

A phosphopeptide is a peptide that contains one or more phosphate groups covalently attached to amino acid residues, most commonly serine, threonine, or tyrosine in eukaryotes. Phosphorylation is carried out by kinases and removed by phosphatases, and it serves as a reversible regulatory modification that can alter a protein’s activity, interactions, localization, and stability.

In cells, phosphopeptides arise as part of signaling and regulatory networks. Because phosphorylation often occurs at substoichiometric levels and the phosphate group can be labile, phosphopeptides are frequently present at low abundance in complex samples. This necessitates selective enrichment before analysis. Common enrichment strategies include immobilized metal affinity chromatography (IMAC) with Fe3+ or Ga3+ and metal oxide approaches such as TiO2, which preferentially bind phosphorylated peptides.

Detection and site localization are typically achieved by mass spectrometry-based proteomics. Tandem MS identifies the peptide sequence and the site of modification, while fragmentation methods vary in their handling of the phosphate group. Electron transfer dissociation (ETD) and related methods can preserve the phosphate during fragmentation, aiding localization, whereas collision-induced dissociation (CID) can cause neutral loss of phosphoric acid, complicating interpretation.

Quantitative phosphoproteomics employs labeling or label-free approaches to compare phosphorylation across conditions, using techniques such as SILAC, iTRAQ, or TMT. Applications range from mapping signaling cascades and cell cycle regulation to biomarker discovery and the study of disease-related phosphorylation patterns. Data on phosphorylation sites are curated in public databases to support reproducibility and cross-study comparisons.