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Title: Characterisation of next generation affinity reagents
Author: Pattison, R.
ISNI:       0000 0004 7428 5299
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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The role of affinity reagents in biological research is essential allowing for the identification, characterisation and functional assignment of proteins. To date, antibodies are the most widely used and studied affinity molecules. The ability for antibodies to bind, with high specificity and high affinity, to the targets they were generated against are features that have been exploited in biological research. However, despite the accomplishments that have been achieved with antibodies, they do possess limitations which have driven the need to develop non-antibody based scaffold proteins that have comparable binding affinity and specificity to antibodies. In addition, exploration of highly complex proteomes, by affinity reagents will enhance our understanding of areas where analytical capabilities are currently limiting findings due to large dynamic range and number of proteins within a given proteome. Affimers, developed as antibody-alternative affinity reagents, are engineered combinatorial proteins possessing three variable interaction sites. This thesis describes the use of mass spectrometry in the characterisation of Affimers and their development for affinity purification mass spectrometry (APMS) workflows. Chapter 3 presents immobilisation strategies for Affimer APMS workflows for the identification of unknown protein binders of naïve Affimers and highlights the associated challenges of nonspecific background binding of proteins and achieving sufficient target enrichment. Four immobilisation methods were assessed and despite the development of an effective method for the enrichment of IgG using Affimers via cysteine-mediated immobilisation, the method was not successful for naïve Affimer target identification. Chapter 4 describes the characterisation of Affimers that target human pepsinogen. Current assays to detect pepsinogen rely on antibodies however they lack any clinical use due to inherent problems with antibodies such as reproducibility and batch-to-batch variability. Five Affimers were expressed, purified and characterised by intact mass spectrometry. As a novel approach to overcome the issue of the large signal of capture reagents in APMS assays, a pepsinogen Adhiron resistant to Lys-C proteolysis was generated. Structural stability of the mutant, assessed by analysing the rate of proteolysis and collision induced unfolding, revealed the mutant exhibits comparable structural stability to the native protein. Chapter 5 presents an alternative approach to Affimer APMS methods for the identification of differentially expressed proteins in sepsis, by using discovery proteomics. Due to the large dynamic range of plasma, an antibody-based spin column depletion method was applied to samples within the study prior to LC-MS/MS analysis. Label-free quantification and bioinformatics analyses of patient cohort 1 and cohort 2 identified 40 and 107 differentially expressed proteins, respectively. A panel of five candidate proteins were selected as potential markers of sepsis and for subsequent Affimer development: CRP, neutrophil-gelatinase associated protein, Protein S100 A8/A9, interleukin-1 receptor-like 1, cathepsin B. Chapter 6 outlines the preliminary development of the major urinary protein (MUP), darcin, as a novel protein scaffold. The disulfide bond in darcin was shown to be vital for providing the high structural stability of the protein, an important feature of protein scaffolds. In addition, preliminary findings demonstrated the development of a darcin resistant to proteolysis may be a suitable approach to overcome the intense signal of the affinity reagent in APMS assays. In summary, this study presents a novel approach to overcome the challenges of APMS with the development of non-digestible protein scaffolds and builds on the literature of common affinity purification contaminant proteins. In addition, the study provides a contribution to sepsis plasma proteome analysis and identifies five proteins implicated in sepsis that provided targets to guide Affimer production for future Affimer immunoassay-based strategies.
Supervisor: Beynon, Robert Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral