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Table 1 Methods for the enrichment of phosphoproteins and phosphopeptides for analysis by mass spectrometry

From: Phosphoproteomics: new insights into cellular signaling

Method

Comments

References

Chemical modification

  

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

  

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]

  1. Abbreviations: P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine.