Mass Spectrometric Methods for the Analysis of Chemically Modified Proteins

Abstract

Mass spectrometry has critical roles in analytical chemistry, biomedical research and the diagnosis and treatment of human disease. Its ability to unambiguously identify and quantify a diverse range of biomolecules, from small molecule metabolites to large intact protein complexes in complex biological matrices, has solidified this technique’s place in the clinical chemists’ laboratory. However, mass spectrometry’s wider adoption by biochemists and clinicians is hindered by the inherent volume and complexity of the data it can generate from a biological context, and the subsequent level of specialist equipment and knowledge required to interpret results in a reliable and meaningful way. It is therefore apparent that a potential solution to this issue is to develop new methodologies, which adapt simple biochemical techniques into mass spectrometry sample preparation workflows that can be performed by biochemists using standard laboratory equipment. This thesis begins by reviewing some recent advances in clinical mass spectrometry and the current limitations of this technology. In doing so, we establish the importance of hyphenated techniques, which merge the concept of immunoassays with mass spectrometric detection to enable its adoption in high-throughput clinical and biomedical research applications. Many of these new approaches are only possible because of the development of efficient biocompatible chemical reactions, which facilitate the covalent modification of antibodies to improve their analysis via mass spectrometry. The concept of chemical labelling with mass tags, or synthetic linkers that fragment during ionisation and/or other stages of gas-phase analysis, has been widely explored as a strategy for detecting large heterogeneous biomolecules, such as antibodies. Chapter 2 explores the design, synthesis and application of an ultra-violet-cleavable linker for labelling intact antibodies to simplify their detection via matrix-assisted laser desorption/ionisation mass spectrometry. The novel aspect of this project is the utilisation of copper-catalysed azide-alkyne cycloaddition, or copper click chemistry, to enable straightforward modification of the detected photodissociation product, thereby offering the potential for multiplexed analysis of biomolecules. Despite the established utility of the copper click reaction, there are several limitations for its application in biological contexts. We therefore sought to identify alternative routes for modifying mass tags. Chapter 3 explores the utilisation of Diels-Alder cycloaddition chemistry for synthesising mass-tagged biomolecules, which conveniently facilitate efficient gas-phase fragmentation, even with the absence of a photocleavable moiety. The ability to easily detect proteins within biological specimens is incredibly valuable for diagnostics; however, the aetiology of many diseases involves modifications to proteins that occur after biosynthesis, or post-translational modifications. Chapter 4 explores the application of bottom-up proteomics methods to identify amino acid modifications to equine heart myoglobin in response to a model for oxidative stress, and characterise the products of its labelling with a novel fluorophore. Whereas the ability to analyse primary protein sequences and post-translational modifications is undoubtedly valuable, the function of many proteins is closely linked to their three-dimensional higher order structures, including transient interactions with other biomolecules. Reagents known as chemical crosslinkers, can be utilised to stabilise interactions between nearby amino acids prior to bottom-up proteomic analysis, thereby retaining tertiary and quaternary protein structural information. Chapter 5 describes some of the recent efforts from our research group to develop efficient strategies for synthesising and applying chemical cross-linkers with improved features for the purification and identification of cross-linked proteins. This thesis therefore explores various potential implementations of mass spectrometry for investigating the dynamic and complex roles of proteins in the context of human disease, from altered expression levels to post-translational modifications and protein-protein interactions.