Phosphoprotein Enrichment by Antibodies and Capture Molecules

Antibodies directed against pSer, -pThr [10], and—pTyr [11] have been used to generally enrich for plant proteins phosphorylated at the respective residues in immuno-precipitation experiments. It should be noted that the use of global antibodies directed against pSer or pThr is usually associated with lower specificity compared to the anti-pTyr antibodies. This might be due to the better accessibility of the phosphate group located on Tyr or because of the lower complexity of the structural nature of the Thr and Ser antigens compared to Tyr. Antibodies directed against regions surrounding a specifically phosphorylated residue can be effectively used to enrich for a certain protein of interest. In addition to antibodies, there are several different protein domains designed by nature, which are capable of binding phosphorylated proteins and peptides. While SH2 (Src Homology 2) and PTB domains recognize predominantly Tyr phosphorylation, 14-3-3 proteins as well as WW domains bind to proteins phosphorylated on Ser and Thr, and FHA domains show a preference toward Thr phosphorylation [12]. In principle, all of these domains or the respective proteins can be used for the enrichment of different sorts of phosphorylated peptides. In fact, immobilized 14-3-3 proteins have been used for the purification of Ser/Thr phosphorylated plant proteins [13, 14]. As described, each of these domains/proteins only recognizes a subset of the total pool of phosphorylated proteins, and experimental setups relying on these domains are therefore not as widely applicable as methods relying on more general affinity mechanisms. A combination of a more general affinity chromatography followed by different more restrictive approaches could, however, further facilitate the analysis of the phosphoproteome.

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