Phosphoproteomic studies have been difficult given the suppression of ionization by the negative charges on phosphate and the non-stoichiometric nature of phosphorylation. To mitigate these problems, we propose eliminating phosphate from proteins using phospholyase enzymes (resulting in dehydroalanine and dehydrobutyrine) followed by the addition of immunogenic probes to allow for separation of previously phosphorylated proteins from cell lysates. A nucleophilic probe to enrich previously phosphorylated serine residues has been developed containing a biotin functionality, and an iodo-functionalized probe has been synthesized to enrich previously phosphorylated threonine residues. Kinetics studies have been performed with the first probe and reactivity of the probe with dehydroalanine has been shown to be enriched 48-fold over dehydrobutyrine. The iodo-functionalized probe has been shown to successfully label both dehydroalanine and dehydrobutyrine. With both of these probes in hand, phosphoproteins will be able to be separated from their nonphosphorylated counterparts and their ionization will be improved since the negative charges on phosphate will no longer be present. This will allow for the discovery of phosphorylation events that are implicated in disease and a better understanding of cellular signaling.

Nucleophilic probes to enrich the phosphoserine and phosphothreonine proteomes

256th National Meeting (2018)

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