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The Proteomic and Quantitative Exploration of N-Mustard Analogues of S-Adenosyl-L-Methionine in Protein Systems

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title
The Proteomic and Quantitative Exploration of N-Mustard Analogues of S-Adenosyl-L-Methionine in Protein Systems
author
Pecor, Lindsay
abstract
Protein methylation of histone and non-histone proteins is a post-translational modification involved in epigenetic and gene expression. The study of protein methylation is challenging due to the small unreactive nature of a methyl group. Many techniques have been developed to aid in the study of these modifications, however, they all have their disadvantages. The development of a more general technique to study methylation would be beneficial to the field of epigenetics. A group of N-mustard S-adenosyl-L-methionine (SAM) analogues have been developed to be used as biochemical probes in DNA and protein systems. Designed as a mimic of SAM, the N-mustard SAM analogues are capable of enzymatic transfer to a target substrate by a methyltransferase. The goal of this work is to demonstrate the utility of the N-mustard SAM analogues in protein systems outside of the model protein arginine methyltransferase 1 (PRMT1)/AcH4-21 model system by utilizing a full-length histone substrate, a non-histone substrate, and the protein lysine methyltransferase,euchromatic heterochromatin methyltransferase 2 (EHMT2). Additionally, this work aims to explore the utility of the N-mustard SAM analogues in more complex environments such as those containing the native methyl donor SAM. The N-mustard SAM analogue was examined in multiple protein systems using the methyltransferases PRMT1 and EHMT2 on both model peptides, histone, and non-histone substrates. Mass spectrometry analysis using the LTQ-Orbitrap XL revealed the site-specific modifications of AcH4-21, histone H4, by PRMT1 without non-specific alkylation and with high sequence coverage using alternative sample preparation techniques. Additionally, the modification of mutant GST-GAR by PRMT1 was confirmed for both methylation and analogue modification. Although methylation was observed at 4 of the 5 assumed sites, analogue was identified at 3 of the sites with only a single modification per peptide detected. Analogue modification of the histone H3 peptide by EHMT2 was also observed, however, confirmation of the exact location of the modification was inconclusive due to low peptide spectrum matches (PSMs) and mass ion coverage. The expanded utility of the N-mustard SAM analogues in the presence of native SAM was explored using an enzyme coupled assay by continuous colorimetric analysis, HPLC, and mass spectrometry analysis with the TSQ Quantum Access MAX triple quadrupole mass spectrometer. Methods were developed for the successful quantitation of methylation through adenine production. However, a decrease in methylation was not observed in the presence of the N-mustard SAM analogue and can be attributed to the use of SAM at saturating concentrations. The work presented here shows the utility of the N-mustard SAM analogues on multiple substrates and their potential as biochemical probes in protein systems.
subject
Epigenetics
Mass Spectrometry
Methylation
Post-Translational Modifications
Proteomics
contributor
Comstock-Ferguson, Lindsay R (advisor)
Hollis, Thomas (committee member)
Dos Santos, Patricia C (committee member)
King, S. Bruce (committee member)
Tracy, Christopher M (committee member)
date
2023-01-24T09:35:50Z (accessioned)
2023-01-24T09:35:50Z (available)
2022 (issued)
degree
Chemistry (discipline)
identifier
http://hdl.handle.net/10339/101778 (uri)
language
en (iso)
publisher
Wake Forest University
type
Dissertation

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