Novel and emerging analytical techniques for the identification and quantification of proteins in complex biological systems
thesisposted on 29.03.2022, 00:32 authored by Michael Mariani
The aim of this thesis was to investigate and further develop a series of novel and emerging techniques in the field of proteomics, used for the identification and quantification of proteins in a range of complex biological systems. This involved pursuing three separate projects all linked by this common theme, as outlined below. -- The first project focussed on testing the viability of a novel chemistry of selfassembled monolayers orientated in concentric circles on MALDI plates (known as a Biochip) and determining if more information for identification of proteins could be obtained utilising such methodologies. I was able to show that the biochips could concentrate simple peptide samples and afford a practitioner 10-100 fold increases in limit of detection in the attomole/μl range compared to standard MALDI methods. I also developed the first hybrid AnchorChip/4800 plate system so that the AnchorChipTM technology could be used in an Applied Biosystems 4800 TOF/TOF mass spectrometer. The biochip did perform similarly to the novel hybrid AnchorChipTM on single protein digests. The ability of the biochip to remove salt contaminants was shown on spiked peptide samples, though the technique was problematic at best on gel plug digests and needs further investigation before it can be considered a viable and robust method. The ability of the biochip to selectively affinity capture and isolate phosphorylated peptides from a protein digest was shown at the femtomole level. However, the biochip lost the ability to concentrate the sample once the new functional chemistry for affinity capture was applied. These results are an interesting proof of concept, but the method still needs further development before it can be considered a working platform that can achieve both affinity capture and concentration of a biological sample mixture. -- The second project was developed to show the potential pitfalls of current bottom-up proteomic methods, namely the misidentification of some proteins in a sample set. The justification for this comes from the protein inference problem and I was aiming to create an argument for the development of better top-down proteomic methods or enhanced bottom-up methods. I developed a novel multidimensional protein fractionation system called PROOF, with a novel graphical interpretation and representation of the peptide data related back to the elution of the proteins from the PROOF system. This highlighted the proof of concept for the application of PROOF to a complex and important proteome such as human plasma, and brought to light truncated or cleaved elements within this proteome that standard bottom-up proteomic analysis could not identify. Specifically, I identified five protein candidates for which I demonstrated new features. This can serve as a basis for future analysis of their endogenous primary structure, as well as possible tertiary and quaternary structural elements. -- The third project involved quantitative proteomics as applied in plant systems. The aim was to develop a sample preparation method that worked in plants for iTRAQ labelling, and compare this with label-free spectral counting methodology in use in our group at the time. The biological aim of this project was to elucidate new information pertaining to the biochemistry of rice under cold stress conditions. I was able to get the iTRAQ labelling to work in a plant system, particularly rice leaf material that had undergone temperature stress. I was also able to show that both quantitative techniques are comparative and identified similar biological insights, while the total number of proteins identified and quantified by spectral counting was proportionately larger, with 236 and 84 proteins for spectral counting and iTRAQ respectively. We were also able to identify two uniquely effected biological pathways for cold stress by spectral counting that iTRAQ did not show; histone production and vitamin B biosynthetic proteins. These results showed that in our hands, spectral counting was more viable than iTRAQ for quantitative proteomic analysis in plant systems. -- The body of work presented in this thesis represents a significant contribution to the field of proteomics. I have developed new approaches, validated existing methods, and used some of these to discover new biological insights - which are the ultimate goal of any proteomics experiment.