Novel Methods for the direct analysis of untreated biological samples using hyphenated ion mobility/ mass spectrometry with ambient ionisation

This project is driven by the need for development of novel analytical technology at AstraZeneca R&D Charnwood, Clinical Pharmacology and DMPK. The technological challenges faced in the investigation of real-world biological samples are centred on the requirement for new methods, which combine the power of emerging techniques to afford increased sensitivity, selectivity and sample throughput. These biological application areas focus upon the determination of biomarkers, peptide and protein fragments, metabolites, protein-bound complexes, and low-molecular weight compounds from a number of complex sample matrices, including plasma, urine, and lung sputum. Conventional mass spectrometry-based analytical methods for biomolecule analysis rely on the use of electrospray (ESI) or matrix-assisted laser desorption/ionization (MALDI) ionization. Whilst these techniques are widely used, both require extensive sample preparation prior to analysis. ESI is susceptible to ion suppression in the presence of salts and other endogenous compounds encountered in biological samples, which must be removed by solid-phase sample extraction prior to LC-MS/MS analysis. MALDI is more tolerant to endogenous material, but requires addition of matrix to the sample. The objective of the proposed studentship is to develop novel analytical tools that allow the direct, robust measurement of biomolecules using desorption electrospray ionization (DESI) combined with ion mobility spectrometry and mass spectrometry (IM-MS). DESI is a new approach to ionization that allows biomolecules to be desorbed directly and rapidly from surfaces without prior sample pre-treatment . Although related to ESI, the DESI technique has been shown to be more robust to the presence of endogenous material. Conventional mass spectrometric platforms, by their very nature, offer little in the way of increasing the selectivity of ion detection, without significant loss of data related to the sampled DESI ion population and separation of the desorbed ions by chromatography is not possible. However, combining DESI with ion mobility (IM) spectrometry and mass spectrometry allows the desorbed ions to be pre-separated prior to mass spectrometric analysis. IM is a gas-phase electrophoretic technique that separates ion on the basis of their charge and collision cross section (i.e. size and shape) on the millisecond timescale. The orthogonal nature of hybrid ion-mobility mass spectrometry (IM-MS) combines electrophoretic with mass-to-charge separation, whilst retaining the DESI-derived ion data. Loughborough University has extensive experience in a number of ion mobility-based techniques and ambient sample ionisation methods, including DESI. The key deliverables of this project are: Deliverable 1: The provision of novel and robust DESI-IM-MS, demonstrating high selectivity and sensitivity for the determination of biomolecules to improve detection and confirmation. Deliverable 2: Reducing sample analysis times from hours to seconds, dramatically increasing sample throughput and removing associated bottlenecks. Deliverable 3: Enable rapid quantitation of biomolecules by spiking untreated samples (isotope dilution, internal standardisation or stable-label isotopes) prior to rapid analysis using DESI-IM-MS. Deliverable 4: Structural and conformational analysis of biomolecules through measurement of collision cross section, allowing greater understanding in wider biological contexts, such as protein conformation and function in a given biological system. Deliverable 5: Combination of the key deliverables above would result in methods inherently transferable to important scientific platforms within the UK biosciences, such as metabolomic and proteomic research, where the sensitive and selective analysis of complex biological samples is paramount.