Insulin resistance is associated with an overall decrease in tyrosine phosphorylation and increased serine phosphorylation of IRS-1. To understand the role that the phosphorylation of IRS-1 serine residues may play in insulin resistance and diabetes we need to i) identify the location of serine phosphorylation sites of IRS-1 and ii) measure the site-specific changes in IRS-1 phosphoserine and phosphotyroserine levels between normal and insulin resistant conditions. Because of the complexity and insensitivity of existing methods for measuring serine phosphorylations, a major aim of this project is to develop a sensitive a robust protocol that is specific to peptides from protein enzyme digests containing serine phosphorylations. Phosphoserine residues will be selectively labeled with either an unlabeled or labeled reagent using a recently report4ed methodology. This modification will then permit the isolation of phosphoserine residues with the addition of a biotin tag and affinity purification. This reagent also contains a cleavable linker to facilitate the release of the enriched peptide from the solid support. During this research training fellowship, I will develop and apply this methodology along with muLC-MS/MS to both locate and quantify the serine phosphorylation sites of IRS-1 associated with insulin resistance.
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