From: Phosphoproteomics: new insights into cellular signaling
Method | Comments | References |
---|---|---|
Chemical modification | Â | Â |
Affinity tagging | Phosphorylated amino acids are derivatized by β-elimination or carbodiimide to introduce tags; β-elimination strategy is limited to P-Ser and P-Thr, and also occurs with O-glycosylated residues. | [46,47] |
Fluorous affinity tagging | Perfluoroalkyl groups are selectively coupled to P-Ser and P-Thr using β-elimination; modified peptides are enriched with fluorous-functionalized stationary phase. This method is highly selective for derivatized peptides. | [48] |
Phosphospecific proteolyis | Chemical modification of P-Ser and P-Thr introduces lysine analogs and cleavage by lysine-specific proteases. This method allows direct identification of phosphorylation site without sequencing the phosphopeptide. | [49] |
Thiophosphorylation and affinity tagging | Proteins are phosphorylated with ATP3S; thiophosphates are alkylated to form linkages with biotin or solid supports. This method requires in vitro phosphorylation and most kinases utilize ATP3S poorly. | [50] |
Bio-orthogonal affinity purification | Analog-specific kinases are used selectively to phosphorylate substrates in vitro or in vivo; the phosphate analogs are then derivatized to generate a hapten for immunoprecipitation. This method requires expression and/or isolation of an engineered kinase. | [51] |
Direct enrichment | Â | Â |
Anti-phosphotyrosine antibodies | Anti-P-Tyr antibodies have proven very useful for the enrichment of P-Tyr-containing proteins; they have been used alone or in combination with IMAC. They have been used to enrich P-Tyr peptides. | [31,33,52,53] |
Anti-phosphoserine and anti-phosphothreonine antibodies | Anti-P-Ser and anti-P-Thr antibodies have been used, but currently are less useful than anti-P-Tyr antibodies. | [54-56] |
Immobilized metal affinity chromatography (IMAC) | Introduction of an esterification step greatly enhances the selectivity of this method, which is very useful, has been widely used and can be automated. | [29,57,58] |
Cation exchange | Strong cation exchange chromatography has been used for the large-scale identification of phosphorylation sites. This method selects for peptides phosphorylated on a single residue. | [34] |