Studies in Protein Post-Translational Modification Using CAD and ETD Mass Spectrometry

Proteins are macromolecules that perform specialized and critical functions for nearly every biological process. Regulation of protein function is required to prevent diseases including cancer. Covalent post-.‐translational modifications (PTMs) can alter protein structure to regulate the activity of many proteins. While several methods are currently used to identify PTMs, mass spectrometry (MS) stands at the forefront because it is an unbiased and sensitive technique that can provide unambiguous identification of the 200+ PTMs currently known. Described within this dissertation are three applications of high resolution MS that implement high performance liquid chromatography, collision-.‐activated dissociation (CAD) and electron transfer dissociation (ETD) to identify novel PTMs on various proteins. First, we describe a comprehensive phosphorylation site analysis of human tensin 1, an extremely large scaffolding protein required for controlled cell migration. By implementing optimized proteolysis, CAD and ETD MS, and phosphopeptide enrichment, we identified an incredible 72 phosphorylation sites on tensin 1. Using differential isotopic peptide labeling with phosphopeptide enrichment, we identified potential protein phosphatase 1α dephosphorylation target sites. This study also revealed regulation by p38α MAPK, a Ser/Thr kinase that extensively phosphorylates tensin 1.

Second, we demonstrate the utility of ETD for the analysis of O-.‐linked N-.‐ acetylglucosamine (O-.‐GlcNAc), a monosaccharide PTM that is notoriously difficult to study using MS. By implementing high resolution MS, ETD and recently developed xxii peptide charge state modification chemistry, we identified 3 novel O-.‐GlcNAc and 15 novel phosphorylation sites on human homeodomain-.‐interacting protein kinase 1.

Third, we present new MS-.‐based methods for the characterization of α-.‐N-.‐ methylation. Although methylation of Lys and Arg side chains has been extensively studied, very few mammalian substrates for α-.‐N-.‐methylation are known. Our new enrichment method incorporates α-.‐N-.‐methylation-.‐specific antibodies, molecular size filtering, high resolution MS and ETD fragmentation. Application of these methods to mouse heart and spleen tissue lysates afforded the identification of several N-.‐terminally methylated proteins that were previously unknown. In addition, we expanded the known N-.‐terminal consensus sequence for mammalian α-.‐N-.‐methylation to include Ala/Pro/Ser-.‐Pro-.‐Lys and Ser-.‐Ser-.‐Lys. As a result, we provided a significant increase in the number of known mammalian α-.‐N-.‐ methylation targets.