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Title: Proteomic applications of protein and peptide transamination
Author: Zhang, H.
ISNI:       0000 0001 2434 4455
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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The extreme amino (N)-terminus of a protein is highly informative of protein stability, localisation, function, as well as regulation. Protein N-termini often undergo posttranslational modifications (PTMs), some of which generate new proteoforms with neo-Ntermini due to proteolytic processing. N-terminal PTMs and the resulting proteoforms greatly contribute to the complexity of the proteome. Additionally, authentic N-termini may differ from those predicted by genome analysis. This thesis describes studies to further develop and to optimise techniques that modify, enrich and identify the N-termini of proteins. The previously reported "NHS-Sepharose technique" removes unwanted peptides after acetylation of amino groups and protease digestion, thereby enriching for N-terminal peptides. Using this negative selection strategy, the desired N-terminal peptides were shown to be contaminated with specific neo-peptides. Various parameters were therefore systematically modified in conjunction with model proteins to achieve complete removal of neo-peptides. Investigation of alternative aminereactive chemistries using N-succinimidyl S-acetylthioacetate or citraconic anhydride indicated that they were not superior to the original amine acetylation approach. The studies also explored a positive selection strategy that directly enriches for protein Ntermini. Selective Transamination Of N-Ends (STONE) is a promising reaction for this purpose. Use of STONE with subsequent biotinylation of the introduced carbonyl groups allowed Nterminal affinity tagging of synthetic peptides and model proteins. It was confirmed that the latter were amenable to avidin-biotin affinity enrichment. Treatment of extracts from Jurkat T-lymphocytes by STONE allowed the experimental verification of N-termini from a range of proteins. Previously unreported N-terminal methionine excision was also uncovered in cytoplasmic serine-tRNA ligase (SYSC_HUMAN) and peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5_HUMAN). STONE-mediated removal of the a-amino groups of peptides and proteins alters their net charge. This suggested that it could be used to achieve orthogonal peptide separations in LCMS/MS, and hence discover additional peptides in shotgun proteomic experiments. Using protein extracts from Jurkat T-cells, STONE was shown to increase the number of identified peptides by 25%, thereby significantly also increasing protein sequence coverage and the number of assigned proteins. The unexpectedly large increase in the number of detected peptides, coupled with the versatility and simplicity of STONE, suggests that it may find widespread use in proteomics.
Supervisor: O'Connor, Carl Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral