Barry L. Karger
Paul Vouros, Graham B. Jones, Roger W. Giese
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Science. Department of Chemistry and Chemical Biology.
chemistry, analytical chemistry, proteins, glycoproteins, liquid chromatography-mass spectrometry (LC-MS), biopharmaceutical
Proteins - Analysis, Mass spectrometry
Amino Acids, Peptides, and Proteins | Biochemistry
This thesis focuses on the development of ultra sensitive high resolution analytical methods for the characterization of proteins and glycoproteins from samples of clinical and biopharmaceutical origin. In the first instance the combination of laser capture micro dissection (LCM) for the selective enrichment of homogenous but low number cell populations in combination with down-stream porous layer open tubular column (PLOT) liquid chromatography-mass spectrometry (LC-MS) using both one- and two-dimensional separations is described. The second portion of the thesis describes the ultra high performance analysis of intact recombinant a-human chorionic gonadotrophin glycoforms using capillary electrophoresis with accurate mass high resolution Fourier transform ion cyclotron resonance mass spectrometry (CE-FTMS).
In Chapter 1 an overview of current analytical methods and technologies applied in the field of proteomics is discussed. A critique of these technologies is also performed laying down the foundations for the developments and improvements in current state-of-the-art as presented in the subsequent Chapters.
In Chapter 2 the development of a micro-proteomic workflow for the comprehensive analysis of just 10,000 cells, collected by LCM, from invasive and metastatic epithelial cell types from a breast cancer patient is described. To minimize sample loss the development of an efficient sampling handling approach was necessary. To achieve this protein level separation and subsequent enzymatic digestion of the cell lysate was performed using short distance SDS-PAGE separation on tricine-PAGE gels. By combining this sample clean-up and fractionation approach with ultrasensitive 1D PLOT LC-MS in excess of 1,000 proteins were identified following injection of just 1/10th of the digested lysate or approximately 1,000 cells. The micro-proteomic workflow is highly suited for the comparative analysis of such small but highly informative LCM collected cell populations, more than 100 proteins were found to be differentially expressed thereby facilitating a deeper understanding of the associated biological changes associated with the invasive to metastatic transition.
In Chapter 3 the application of an online 2D-RP/SCX/SPE/PLOT LC-FT-MS micro-proteomics platform is presented for the comparative proteomic analysis of LCM collected normal and triple negative breast cancer cell population. Using the effective sample handling approach described in Chapter 2 followed by fractionation and ultra sensitive analysis of the lysate, the tryptic digest corresponding to 4,000 cells using the 2D-RP/SCX/SPE PLOT LC-FT-MS platform in excess of 15,000 unique peptides corresponding to 4,259 proteins were identified. This deep proteome coverage further emphasizes the utility of the developed micro-proteomic platform for the analysis of trace quantities of proteins generated from small but highly biologically important LCM enriched cell populations.
In chapter 4 the development and application of a high resolution CE-FTMS method for intact glycoform profiling of recombinant á-human chorionic gonadotrophin is described. The CE separation parameters used allowed for the rapid analysis,minutes, and high resolution of >60 different glycoforms bearing up to nine sialic acids in addition to other glycoforms differing by the number and extent of uncharged monosaccharides. A low volume pressurized liquid junction, which preserves the high resolution of the CE separation, was used to interface the CE system with high resolution FTMS thereby allowing accurate determination of charge state and accurate mass of each intact glycoform following deconvolution. In addition to the intact glycoform, profiling analysis of glycopeptides and glycans was also performed to determine and assign the population of oligosaccharides present at each individual glycosite, thereby facilitating complete and comprehensive characterization of r-ahCG. The methodology developed in Chapter 4 was further applied to the analysis of r-áhCG from different expression systems, CHO and murine cell based. The CE-FTMS method is readily applicable for characterization of drug substance/product as well as in process monitoring of these complex glycoforms.
Dipak A. Thakur
Thakur, Dipak A., "Development of sensitive high performance analytical methods for the comprehensive characterization of proteins and glycoproteins from samples of clinical and biopharmaceutical importance" (2011). Chemistry Dissertations. Paper 30. http://hdl.handle.net/2047/d20001090
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