Leveraging Intact, Limited Enzymatic Digestion, and Specific Enzymatic Digestion Approaches with Liquid Chromatography Tandem Mass Spectrometry for Protein Analysis

LC-MS/MS analysis is a powerful tool for proteomic studies; however, there is not a single analysis method that is universal for all proteomic questions or challenges. Continued development and optimization LC-MS/MS analysis methods enable the tailoring of methods to answer specific biological questions. The work in this dissertation details the LC-MS/MS analysis of three different classes of proteins and the specific methods used for each.
The first biological question addressed is how the development of sperm nuclear basic proteins differs in various species. To answer this question, proteins in complex cell lysate samples must be identified in different species. Hydrophilic liquid chromatography coupled with high resolution MS/MS intact protein analysis was used for the sequence characterization of sperm nuclear basic proteins in arthropods. Insect sperm sample analysis yielded 23 novel proteins in four different insect species: Parachauliodes continentalis (fishfly), Apis mellifera (honeybee), Nasonia vitripennis (jewel wasp), and Graptopsaltria nigrofuscata (large brown cicada). The arachnid sperm sample, Streada nobilis (noble false widow spider), analysis yielded detection and partial sequence identification of 2 sperm nuclear basic proteins.
The antibody class of proteins is currently used as treatments for of diseases such as cancer and autoimmune diseases. However, for an antibody to succeed as a treatment with minimal side effects, all post-translational modifications must be identified. Limited digestion of antibodies produces large peptides that overlap with each other for LC-MS/MS analysis. Limited digestion with a capillary immobilized enzyme reactor is a powerful tool that enables antibody sequence and post-translational modification identification. However, the current method is not accessible to most researchers and not applicable to high throughput analysis. This dissertation details the development of a micro-immobilized enzyme reactor that was used to digest a globular protein and a monoclonal antibody to enable sequence analysis. Digestion by a micro-immobilized enzyme reactor produced unique peptides that overlapped to provide full amino acid sequence identification. This method required limited training and sample preparation, and the capablity for the digestion of up to 20 proteins in 30 minutes for LC-MS/MS analysis.
Researchers, interested in using glutamylation as a drug target for acute myeloid leukemia, aimed to identify a change in glutamylation of wild-type nucleophosmin 1(NPM1) and mutated NPM1c in acute myeloid leukemia cells. The mutated NPM1c, found only in some types AML cancers, has been linked to leukemogenic impacts; however, the exact biological mechanism is not understood. LC-MS/MS analysis enabled the identification of both wildtype NPM1 and mutated NPM1c in recombinant protein and AML cellular samples. Specific enzymatic digestion enabled site localization of glutamylation in cellular samples and the change in glutamylation between each of five protein samples. The increased glutamylation identified in samples containing NPM1c provide information that could enable improved drug targeting for AML treatment.