Kotowska, Anna
(2022)
Enabling in situ protein analysis for pharmaceutical applications using 3D OrbiSIMS.
PhD thesis, University of Nottingham.
Abstract
In situ characterisation of proteins at surfaces is of interest to biological, biotechnological and pharmaceutical fields, specifically areas of research such as tissue imaging, biomaterials discovery, biosensor development and tissue engineering. Label-free protein characterization at surfaces is commonly achieved by matrix assisted laser desorption ionisation (MALDI) and requires digestion and/or matrix deposition prior to mass spectrometry, limiting the spatial resolution in two and three dimensions. This work presents the use of secondary ion mass spectrometry, 3D OrbiSIMS, to achieve the first matrix-, digestion- and label-free assignment of proteins in solid three-dimensional samples.
Primary ion beam-induced fragmentation of undigested proteins resulted in spectra containing characteristic fragment ions, which can be used for de novo sequencing protein identification. This approach, termed ballistic sequencing, enabled amino acid sequence assignment covering from 5 to 53% of the sequence in 16 analysed proteins. The similarities and differences from classical proteomic methods were established and features such as proton, ligand or cell-binding sites were detected in the analysed proteins.
The method proved to be sensitive enough to detect highly specific protein fragments in the 3D OrbiSIMS spectra from a protein monolayer sample, equating to 40 femtomoles of a protein per analysis. This demonstrates the unique capability of ballistic sequencing to characterise protein surfaces, when the amount of analyte is too small to be extracted from the surface for analysis via existing methods such as MALDI. To further test the method, three example proteins were mapped throughout the depth of a complex biological sample, human skin. Collagen, keratin and corneodesmosin were detected in the dermis, epidermis and stratum corneum, respectively, which is consistent with known distribution of these proteins in skin.
A reproducible method of manufacturing monodisperse PLGA micro- and nanoparticles was successfully developed for subsequent incubation with biological media and investigation of protein adsorption on the surface. A reliable size tuning in a range between 120-250 nm and 5-20 μm was achieved by the use of droplet microfluidics and modifying parameters such as the polymer concentration and the flow rate of continuous and dispersed phases.
The polymeric particles were incubated with serum albumin and whole serum and analysed in situ using cryogenic capabilities of the 3D OrbiSIMS. The chemical image of particle surface was not achieved, however the preliminary results obtained in this work, such as detection of characteristic serum albumin peaks in the frozen particles sample, indicate that further method development could enable use of the 3D OrbiSIMS as an emerging technique for in situ analysis of particles’ interaction with biological media.
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