The use of recently developed mass spectrometry approaches for the characterisation of biological mixtures

The thesis describes a number of examples of the use of recently developed mass spectrometry experimental approaches to characterise biologically important mixtures. The recently introduced field of ambient ionisation mass spectrometry has been utilised in the rapid, sensitive, information rich characterisation of pharmaceutical formulations. Little, or no, sample treatment was required and the experiments were shown to provide detailed information on active ingredients in the presence of a number of other components. A number of ambient ionisation approaches including DART, DESI and DAPCI were compared and advantages and disadvantages of each approach outlined and discussed. The exciting technology of ion mobility has recently been commercially interfaced with mass spectrometry (IMMS). This has been utilised in a series of fundamental experiments that probe the interaction of varied cations with isomeric oligomers of carbohydrates. The approach enables conformational changes to be rapidly measured over a wide (500-6000 Da) mass range. Changes in conformations were observed for multiply cationised species which agree with previously measured solution phase measurements. The IMMS approach has also been used successfully to characterise a number of Nlinked glycans released from glycoproteins. The experiments enable isomeric structures to be differentiated and present an opportunity to develop a rapid, high information content screen. Estimated cross sectional measurements have been calculated and found to be in good agreement with those obtained from conventional drift cell approaches

The use of recently developed mass spectrometry approaches for the characterisation of biological mixtures

The thesis describes a number of examples of the use of recently developed mass spectrometry experimental approaches to characterise biologically important mixtures. The recently introduced field of ambient ionisation mass spectrometry has been utilised in the rapid, sensitive, information rich characterisation of pharmaceutical formulations. Little, or no, sample treatment was required and the experiments were shown to provide detailed information on active ingredients in the presence of a number of other components. A number of ambient ionisation approaches including DART, DESI and DAPCI were compared and advantages and disadvantages of each approach outlined and discussed. The exciting technology of ion mobility has recently been commercially interfaced with mass spectrometry (IMMS). This has been utilised in a series of fundamental experiments that probe the interaction of varied cations with isomeric oligomers of carbohydrates. The approach enables conformational changes to be rapidly measured over a wide (500-6000 Da) mass range. Changes in conformations were observed for multiply cationised species which agree with previously measured solution phase measurements. The IMMS approach has also been used successfully to characterise a number of Nlinked glycans released from glycoproteins. The experiments enable isomeric structures to be differentiated and present an opportunity to develop a rapid, high information content screen. Estimated cross sectional measurements have been calculated and found to be in good agreement with those obtained from conventional drift cell approaches.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The application of capillary electrophoresis and mass spectrometry to clinical and environmental problems

Using capillary electrophoresis (CE) as a separation technique has allowed analytes, previously difficult to separate by standard methods because they did not conform to requirements for GC or HPLC, to be separated with speed and great efficiency. The only requirements for CE analysis are that the sample is soluble in a liquid matrix and that analytes are present as positive or negative ions whilst in this matrix. This technique has been used here to analyse both clinical and environmental samples, some as cations and others boron-containing complexes as anions. Samples were analysed using a combination of CE alone, mass spectrometry alone and also coupled capillary electrophoresis/electrospray mass spectrometry (CE/ES). Clinically orientated analytes, dipeptides in urine and acylcamitines from blood spots were examined and peaks detected directly via uv absorbance. The environmental samples analysed included those which contained chromophoric or non-chromophoric herbicides as well as those containing diisocyanates. Analytes were either detected in their native form as with the dipeptides and chromophoric herbicides, or more typically after derivatisation to improve their absorbance characteristics. The exception was the non-chromophoric herbicides which were detected via indirect uv. CE was an experimental technique for the analysis of all these compounds, except for the dipeptides, all the others having originally been analysed using HPLC or GC methods. In each case an evaluation of the CE method was performed to determine the suitability of the method. By analysing standards in each case, it was possible to confirm that the technique was suitable for qualitative and quantitative analysis of each class of compound. CE proved to be a viable technique for the separation of all classes of compound dealt with in this thesis. However the method could not be relied upon to confirm the identity of these analytes by their migration time alone. To identify the analytes, experiments were carried out to couple CE with a mass spectrometer. Two techniques of mass spectrometry were used within this thesis, fast atom bombardment and electrospray but only electrospray ionisation mass spectrometry was used to couple to capillary electrophoresis and was the only mass spectrometric technique used to analyse clinical and environmental samples. CE instruments were successfully coupled to an electrospray mass spectrometer which then became the detector. Mass/charge ratio measurements were obtained for each analyte used and these allowed the unambiguous identification of each analyte. Other work involved using CE, ES and FAI3 mass spectrometry, to develop a new technique to detect diol containing compounds. This involved complexing the diol with a boron-containing acid to produce an anion which could then be detected using ES and FAB mass spectrometric methods. This work was viewed as a possible technique for the detection of diol containing lipids found within some body fluids

Phosphoproteomics Analyses to Identify the Candidate Substrates and Signaling Intermediates of the Non-Receptor Tyrosine Kinase, SRMS

SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylaton sites) is a non-receptor tyrosine kinase that belongs to the BRK family kinases (BFKs) and is evolutionarily related to the Src family kinases (SFKs). Like SFKs and BFKs, the SRMS protein comprises of two domains involved in protein-protein interactions, namely, the Src-homology 3 domain (SH3) and Src-homology 2 domain (SH2) and one catalytic kinase domain. Unlike members of the BFKs and SFKs, the biochemical and cellular role of SRMS is poorly understood primarily due to the lack of information on the substrates and signaling intermediates regulated by the kinase. Previous biochemical studies have shown that wild type SRMS is enzymatically active and leads to the tyrosine-phosphorylation of several proteins, when expressed exogenously in mammalian cells. These tyrosine-phosphorylated proteins represent the candidate cellular substrates of SRMS which are largely unknown. Further, previous studies have determined that the SRMS protein displays a characteristic punctate cytoplasmic localization pattern in mammalian cells. These SRMS cytoplasmic puncta are uncharacterized and may provide insights into the biochemical and cellular role of the kinase. Here, we utilized mass spectrometry-based quantitative label-free phosphoproteomics to (a) identify the candidate SRMS cellular substrates and (b) candidate signaling intermediates regulated by SRMS, in HEK293 cells expressing ectopic SRMS. Specifically, using a phosphotyrosine enrichment strategy we identified 663 candidate SRMS substrates and consensus substrate-motifs of SRMS. We used customized peptide arrays and performed the high-throughput validation of a subset of the identified candidate SRMS substrates. Further, we independently validated Vimentin and Sam68 as bonafide SRMS substrates. Next, using Titanium dioxide (TiO2)-based phosphopeptide enrichment columns, we identified multiple signaling intermediates of SRMS. Functional gene enrichment analyses revealed several common and unique cellular processes regulated by the candidate SRMS substrates and signaling intermediates. Overall, these studies led to the identification of a significant number of novel and biologically relevant SRMS candidate substrates and signaling intermediates, which mapped to a number of cellular and biological processes primarily involved in cell cycle regulation, apoptosis, RNA processing, DNA repair and protein synthesis. These findings provide an important resource for future mechanistic studies to investigate the cellular and physiological functions of the SRMS. Studies towards characterizing the SRMS cytoplasmic puncta showed that the SRMS punctate structures do not colocalize with some of the major cellular organelles investigated, such as the mitochondria, endoplasmic reticulum, golgi bodies and lysosomes. However, studies investigating the involvement of the SRMS domains in puncta-localization revealed that the SRMS SH2 domain partly regulates this localization pattern. These results highlight the potential role of the SRMS SH2 domain in the localization of SRMS to these cytoplasmic sites and lay important groundwork for future characterization studies