Nucleophilic Probes to Enrich the Phosphoserine and Phosphothreonine Proteomes.
Abularrage, Nile S.
2018
- Phosphorylation is an important posttranslational modification to proteins that enables regulation of protein activity as well as signaling via cascades of kinases. Phosphoproteomic studies have been difficult given the suppression of ionization by the negative charges on phosphate and the non-stoichiometric nature of phosphorylation. Phosphotyrosine has been able to be studied on a proteomic scale ... read moresince there are effective antibodies against it, but all antibodies that have been developed against phosphoserine and phosphothreonine have been sequence specific. 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. In addition to enrichment, this will also increase ionization. A nucleophilic probe to enrich previously phosphorylated serine residues has been developed containing a biotin functionality, and an iodo-functionalized biotin derivative has been developed to target dehydrobutyrine using radical chemistry. Kinetics studies have been performed with the nucleophilic probe and it has been shown to be selective for dehydroalanine. The radical reaction has been shown to successfully label both dehydroalanine and dehydrobutyrine. With both of these probes in hand, phosphoproteomic analysis will be improved by enrichment using the immunogenic handles on the probes and ionization will also be improved since the previously phosphorylated site will no longer contain negative charges. To this end, preliminary lysate experiments have been performed that show that phospholyase enzymes can successfully eliminate phosphate in cell lysates and future experiments will show that the synthesized probes can label the eliminated sites in lysates.read less
- ID:
- bk128n82p
- Component ID:
- tufts:sd.0000773
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- TARC Citation Guide EndNote
