High throughput surface mass spectrometry-based proteomics & metabolomics for biological applications

Meurs, Joris (2021) High throughput surface mass spectrometry-based proteomics & metabolomics for biological applications. PhD thesis, University of Nottingham.

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Surface-based mass spectrometry analysis benefits from the minimum sample preparation required and high throughput nature of the analysis (few minutes per sample) and shows therefore potential for tackling current issues in the field of proteomics and metabolomics. This thesis aims to develop a robust high throughput methodology for the quantitative analysis of surface-adsorbed proteins and for untargeted metabolomics

One of the current problems in the field of biomaterials research is the limited understanding of the mechanistic behind cell attachment and behaviour on polymeric substrates. Fully synthetic substrates have been identified which support growth and survival of human pluripotent stem cells. Pluripotent stem cells are a valuable cell type for regenerative medicine due to their ability to differentiate into the three germ layers. To treat a single patient, more than a billion stem cells are required. Current cell systems use biological feeder layers for stem cell expansion. However, these animal-derived matrices are expensive, undefined, and show high batch-to-batch variation. In order to move towards reproducible, industrial culturing of stem cells a suitable growth substrate needs to be defined. Through high throughput biomaterials discovery, it was shown that some fully synthetic polymers can maintain stem cell cultures to a similar level as biological substrates.

Current understanding of the response of cells on those synthetic polymers is relatively poor. Research has shown that coating of synthetic polymers with culture medium-derived proteins increase the cell attachment which is required for cell survival. This shows the potential role of culture medium proteins in the response mechanism of cells on synthetic polymers. However, current technology does not allow analysis of (combinatorial) polymer libraries which has limited the understanding of relation between cell response and physicochemical properties and molecular features of the polymers. A full understanding of the cell-polymer response mechanism would allow the development and rationalisation of synthetic polymers for culturing of pluripotent stem cells.

It was shown that liquid extraction surface analysis-tandem mass spectrometry (LESA-MS/MS) is a suitable analytical technique for the analysis of in situ digested proteins. LESA is a commercial system which can automatically extract analytes from a given substrate and directly introduce the sample in to the MS. Here, this potential was further explored for polymer array screening as well as polymers taken forward for scale-up experiments. A suitable substrate was chosen (Droplet Microarray) which allowed control over the spreading of the monomer solutions, digestion solution, and organic extraction solvent for reproducible MS results. With carefully optimised LESA and MS parameters, difference in protein adsorption could be detected between different chemical surfaces. These difference in protein adsorption did not show a good correlation with the observed cell response (attachment and number of pluripotent stem cells). Through multivariate modelling was found that surface chemistry was found to play a role in protein adsorption. Whilst array screening did not reveal solid evidence of the importance of protein adsorption in relation to cellular response, experiments of protein adsorption on a larger surface area (6-well plates) revealed higher protein adsorption on polymers with higher numbers of pluripotent stem cells. Altogether, LESA-MS/MS shows to be an interesting tool to quantitatively assess protein adsorption on synthetic polymers. The developed methodology can not only be further used to study more complex growth media for human cell lines, but also extended study the relation between protein adsorption and response of different organisms. The addition of LESA-MS/MS to high throughput screening of material microarrays might reveal vital information and could assist in proper choice of polymers for biomedical purposes.

Further interest of surface analysis comes from the field of oncometabolomics. In this thesis, paediatric ependymoma were analysed by Orbitrap secondary ion mass spectrometry (3D OrbiSIMS) and LESA-MS/MS. The main challenge here was to acquire data using only minimal tumour tissue which was available in the form of a tumour tissue microarray. By analysing the same tumour tissue with two complementary mass spectrometry techniques, a more complete metabolite profile could be obtained. Moreover, the combination of 3D OrbiSIMS and LESA-MS/MS data followed by partial-least squares discriminant analysis (PLS-DA) permitted the classification of tumour tissue based on eventual recurrence. This means that certain metabolite levels are indicative of tumour relapse. Understanding these changes in metabolite abundance along with the changes in corresponding metabolic pathways could open new insight into ependymoma relapse. Further, this analytical strategy would be suitable to study other types of (tumour) tissues.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Kim, Dong-Hyun
Alexander, Morgan R.
Barrett, David A.
Keywords: Surface-based mass spectrometry, Biological applications, Metabolomics, Proteomics
Subjects: Q Science > QD Chemistry > QD 71 Analytical chemistry
Faculties/Schools: UK Campuses > Faculty of Science > School of Pharmacy
Item ID: 65689
Depositing User: Meurs, Joris
Date Deposited: 31 Dec 2021 04:40
Last Modified: 31 Dec 2021 04:40
URI: http://eprints.nottingham.ac.uk/id/eprint/65689

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