Phosphoproteomes

Phosphoproteomes are the complete set of phosphorylated proteins in a cell, tissue, organism, or biological sample at a given time. Phosphorylation, the covalent addition of a phosphate group to serine, threonine, or tyrosine residues, modulates protein activity, localization, interactions, and stability. The phosphoproteome is highly dynamic and condition-dependent, reflecting cellular signaling states such as cell cycle progression, stress responses, or developmental cues. Global phosphoproteomics aims to identify and quantify phosphorylation sites across the proteome to illuminate signaling networks.

Most phosphoproteomic analyses rely on mass spectrometry following enrichment of phosphopeptides due to their low abundance. Common enrichment methods include immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) using TiO2 or Fe3+. After enrichment, liquid chromatography-tandem MS (LC-MS/MS) identifies phosphopeptides and assigns phosphorylation sites. Quantitative approaches such as label-free quantification, iTRAQ/TMT, and SILAC enable comparison across conditions. Site localization scores estimate confidence in the exact phosphorylated residue.

Phosphoproteomics informs the mapping of kinase-substrate relationships, signaling pathways, and cellular decision-making processes. It supports studies in cell cycle control, development, immunology, and disease, including cancer where aberrant phosphorylation patterns are common. Data integration with protein interaction networks and motif analyses aids kinase prioritization and network reconstruction.

Challenges include the substoichiometric and transient nature of phosphorylation, sample complexity, and ambiguity in site localization. Phosphosite obscurity and dynamic range limitations require careful experimental design. Emerging approaches aim at single-cell or spatial phosphoproteomics, which are technically demanding but increasingly feasible.

Public resources curate phosphosite data, such as PhosphoSitePlus, PhosphoGRID for model organisms, and UniProt annotations; large-scale phosphoproteome datasets are deposited in repositories like PRIDE, enabling meta-analyses and pathway mapping.