Protein phosphorylation refers to the process by which a protein transfers the phosphate group on ATP or GTP to a specific site (amino acid residue Ser, Tyr, Thr) on a protein under the catalysis of phosphorylase. Phosphorylation is a widely occurring post-translational modification, with over 30% of cellular proteins undergoing phosphorylation. Therefore, phosphorylation is the most fundamental, widespread, and important mechanism for regulating and controlling protein activity and function. Phosphorylation is involved in various physiological and pathological processes, regulating cellular proliferation, development, differentiation, apoptosis, and other life activities, and is widely applied in research fields such as signal transduction pathways, apoptosis, development and differentiation, and cancer mechanisms.

Phosphorylation proteomics aims to identifying phosphorylated proteins and peptides in diverse samples, accurately localizing and quantifying phosphorylation sites, and ultimately elucidating their biological functions. This methodology begins with protein sample digestion into peptide mixtures, followed by liquid chromatography to separate digested peptide components and reduce sample complexity. Subsequently, phosphorylation peptides are enriched using high-quality phosphorylation modification antibodies and biological materials. Finally, mass spectrometry technology with DDA (data-dependent acquisition) mode is employed for analysis and quantification.