The development and application of novel methods for top-down and structural proteomics
21 October 2016
UAntwerp, Campus Groenenborger, U0.25 - Groenenborgerlaan 171 - 2020 Antwerpen (route: UAntwerpen, Campus Groenenborger
Organization / co-organization:
Department of Chemistry
Frank Sobott & Dirk Valkenborg
PhD defence Frederik Lermyte - Faculty of Science, Department of Chemistry
Virtually every process occurring in living organisms involves the action of proteins. These linear biopolymers, although composed of a limited set of twenty amino acids, adopt a variety of three-dimensional structures, and this higher-order structure is crucial to their function. A second aspect of higher-order protein structure involves the protein’s interaction partners and the architecture of the resulting complexes. As a ‘malfunction’ at any of these stages often results in disease and/or death, there is obviously a significant need for analytical techniques which (1) are sensitive to multiple (preferably all) levels of protein structure, (2) are widely applicable, and (3) require a minimal amount of time and sample preparation.
Mass spectrometry (MS) has for some time been the method of choice for rapidly determining the primary structure (i.e. amino acid sequence) of proteins. However, while a change in primary structure can, and often does, result in changes in higher-order structure, conventional workflows are not inherently sensitive to 3D structure, i.e. two different ‘shapes’ of the same protein will exhibit identical behavior.
By careful control of both solution and gas-phase parameters, it is possible to transfer intact proteins into the vacuum of a mass spectrometer whilst preserving much of the biologically relevant 3D structure. This approach is referred to as ‘native’ mass spectrometry. In this thesis, we demonstrate the use of native MS, in conjunction with two other novel technologies, electron transfer dissociation (ETD) and ion mobility spectrometry, to obtain information about (changes in) higher-order protein structure in the gas phase beyond that revealed by conventional MS experiments.
In this work, we show how ETD can provide information about the exposed surface and unfolding pathways of large, noncovalent protein complexes. Furthermore, we show how certain side reactions occurring under ETD conditions can be promoted, allowing for simplification of ‘crowded’ mass spectra. We have also contributed to the development of software to process the enormous quantities of data generated by MS experiments. This includes a method of accurately (with deviations normally around 0.0001%) estimating the (typically not directly observed) monoisotopic mass of an intact protein, with the potential for facilitating automated matching of experimentally detected proteins with database information. Furthermore, a different algorithm allows for rapid quantification of the relative probabilities (and therefore rates) of different reaction channels available to a protein ion under ETD conditions. In the course of this work, shape-dependent depletion of certain reaction products, measurable by this software, was discovered. As such, it was shown that this reaction pathway analysis provides a novel ‘window’ into (changes in) gas-phase structure of protein ions.