Towards the absolute quantification of protein isoforms through the use of stable-isotope dilution mass spectrometry

Kelly, Robert Noel (2013) Towards the absolute quantification of protein isoforms through the use of stable-isotope dilution mass spectrometry. PhD thesis, University of Glasgow.

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While the existence of protein was first described by Berzelius and Mulder back in 1838 and a single empirical formula noted (C400H620N100O120P1S1) (Vickery, 1950, Brand, 1946), early protein-based research was limited to the analysis of proteins which could be easily purified in large quantities, such as those obtained from blood, egg whites and those obtainable from slaughterhouses, such as digestive and metabolic enzymes (Chapman, 2005). Indeed, despite the development of recombinant deoxyribonucleic acid technologies in the 1970s (enabling protein expression) and the increasing sensitivity of techniques which enable the identification and sequencing of proteins separated by gel electrophoresis (Patterson and Aebersold, 2003), it was not until the late 1980s, with the description of soft biomolecule ionisation that large scale proteomic analyses were undertaken, based upon the use of mass spectrometry (Guerrera and Kleiner, 2005).

While early mass spectrometry-based proteomic analyses focussed on the systematic identification of a great number of proteins within a single organism, the field of proteomics is now becoming increasingly quantitative (Baak et al., 2005), enabling the relative comparison of protein expression patterns between phenotypes, but also the targeted absolute quantification of specific proteins.

During this project, a stable isotopically labelled internal standard based absolute quantitative technique, first described by Gerber and co-workers in 2003 (S. A. Gerber et al., 2003), was applied to the absolute quantification of three families of multiple protein isoforms. This area of research is of particular scientific interest as it is thought that up to 95% of human multi-exon genes may be subject to alternative splicing, making alternative splicing the rule, not the exception (Pan et al., 2008a). Indeed alternative splicing has also been implicated as both a cause and a consequence of disease. This technique should therefore enable both the confirmation of disease, based upon the identification of a set of phenotype specific protein biomarkers, but also the mapping of a disease’s progression (Venables, 2004).

During this study, stable isotopically labelled internal standard peptides were selected for the absolute quantification of 11 confirmed protein isoforms, and two predicted protein isoforms. In addition, a separate MRM based LC-MS acquisition method was developed for the absolute quantification of each of the three families of protein isoforms (A-Raf, PDE4B and SERCA2) within a single analysis, and finally, these acquisition methods were applied to the absolute quantification of a range of immunoprecipitated, exogenously expressed protein isoforms. This project was, however, hindered by the sensitivity of the mass spectrometers available for use, preventing these acquisition methods from being applied to the absolute quantification of the endogenous levels of protein expression.

While beyond the scope of this project, the further development of this quantitative technique should enable future researchers to: (i) Quantify each endogenously expressed protein isoform within a family of multiple protein isoforms. (ii) Assess any changes in the expression of each isoform in a range of cellular states, and (iii) Assess how a targeted drug treatment may affect the expression ratio of these protein isoforms.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Protein quantification, protein quantitation, mass spectrometry, LC-MS/MS, AQUA, A-Raf, SERCA, PDE4B
Subjects: Q Science > QD Chemistry
Q Science > QH Natural history > QH301 Biology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Supervisor's Name: Baillie, Dr. George and Morrice, Dr. Nick
Date of Award: 2013
Depositing User: Dr R N Kelly
Unique ID: glathesis:2013-4401
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 02 Jul 2013 08:41
Last Modified: 02 Jul 2013 08:41

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