Thus far this approach has identified in addition to known PP-1C binding proteins several potential novel subunits and substrates. Several novel sequences have also been obtained including those belonging to a protein kinase that co-purifies through out our the affinity procedure. In collaboration with Dr Timothy MacDonald, we have greatly expended our original idea of modifying phospho amino acids on protein and peptides. Recent advances in the technology include the development of solid state derivatization that allows both selective modification of individual phosphoamino acids and enrichment of the sample. Currently, the technology works well on phosphopeptides.
Some work is required before it can be applied directly to proteins.
We are currently applying the technology to measure changes in protein phosphorylation in samples derived from animals and humans. In the human study, muscle biopsy samples are taken from normal fasted males treated plus minus insulin. The tissue is extracted and proteins separated by 2D SDS-PAGE, then blotted onto Gortex membrane. The released peptides are reacted with the fluorochemistry and phosphates tagged. Following the normal study, we plan to examine the effects of type II diabetes on the phosphorylation state of insulin targets in vivo.