Leveraging Proteomic Strategies to Analyze Post-Translational Modifications and Membrane Proteins

This dissertation details three projects that apply tandem mass spectrometry to analyze purified proteins. There are three strategies that can be used to approach proteomic analysis: intact, limited digestion, and traditional digestion. All three of these projects are unified in that at least two of these strategies was required to achieve their goal. This dissertation demonstrates that these approaches are much more powerful and provide more information in combination than they do alone.

The first project identified a single type of post-translational modification, glutamylation, in a variety of samples derived from recombinant Nucleosome Assembly Protein 1 (Nap1). Previous work found endogenous Nap1 to be extensively modified in the A1 and A3 acidic regions. The goal of this project was to determine if recombinant Tubulin Tyrosine Ligase Like protein 4 (hsTTLL4) was capable of glutamylating in vitro. Using the intact approach, full length Nap1 was readily identified as extensively glutamylated. The traditional digestion approach provided more detail as to the location and extent of glutamylation. This work showed that hsTTLL4 is capable of glutamylating the A1 and A3 acidic regions as single peptides as well as in the context of recombinant full-length Nap1. Further work is required to optimize the incubation of hsTTLL4 with the peptide and Nap1 constructs to achieve similar glutamylation states to endogenous Nap1.

The second project extended the application of a nonspecific enzyme reactor toward membrane proteins. Despite their biological importance, these proteins are insufficiently studied by mass spectrometry primarily because of their hydrophobicity and low charge density. Membrane proteins often require harsh detergents to remain soluble in aqueous solutions and are not efficiently digested by specific proteases. Since the enzyme reactor is not sequence dependent and is already known to be capable of digestion under relatively denaturing conditions (8M urea), it may be the ideal system for analyzing membrane proteins. This chapter broadens the utility of the reactor by determining the stability of the reactor in a variety of buffers commonly used to solubilize membrane proteins. Once the stability of the reactor under these conditions was understood, this knowledge was applied to a small, alpha-helical membrane protein, where progress was made toward complete sequence coverage. By combining sequence coverage from the trypsin and enzyme reactor digestions, nearly 80% sequence coverage of a small membrane protein was achieved.

The final project identified S-acylation on a single pass membrane protein, p75. Acylation is a long-fatty acid modification that is notoriously difficult to analyze due to the hydrophobicity and instability of the modification, which are only exacerbated by the issues of handling membrane proteins. Three sequential specific protease digestions and a nonspecific enzyme reactor digestion were required to obtain high sequence coverage of this single pass membrane protein. Additionally, a mass spectrometry friendly detergent, Invitrosol, was found to be crucial for the identification of fatty acid modified peptides. Although previous work showed p75 was palmitoylated at a single cysteine, this work identified three sites of modification by both fully saturated palmitoyl and monounsaturated palmitoleoyl. This chapter reports the first time palmitoylation has been identified on p75 by mass spectrometry and the first time palmitoleoylation has been identified on p75.