Proteomic analysis of liver membranes through an alternative shotgun methodology
thesisposted on 2022-03-28, 02:13 authored by Joel Chick
The aim of this thesis was to develop a proteomics methodology that improves the identification of membrane proteomes from mammalian liver. Shotgun proteomics is a method that allows the analysis of proteins from cells, tissues and organs and provides comprehensive characterisation of proteomes of interest. The method developed in this thesis uses separation of peptides from trypsin digested membrane proteins by immobilised pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two dimensional shotgun proteomics. In this thesis, peptide IPG-IEF was shown to be a highly reproducible, high resolution analytical separation that provided the identification of over 4,000 individual protein identifications from rat liver membrane samples. Furthermore, this shotgun proteomics strategy provided the identification of approximately 1,100 integral membrane proteins from the rat liver. The advantages of using peptide IPG-IEF as a shotgun proteomics separation dimension in conjunction with label-free quantification was applied to a biological question: namely, does the presence of a spatially unrelated benign tumor affect the abundance of mouse liver proteins. IPG-IEF shotgun proteomics provided comprehensive coverage of the mouse liver membrane proteome with 1,569 quantified proteins. In addition, the presence of an Englebreth-Holm-Swarm sarcoma induced changes in abundance of proteins in the mouse liver, including many integral membrane proteins. Changes in the abundance of liver proteins was observed in key liver metabolic processes such as fatty acid metabolism, fatty acid transport, xenobiotic metabolism and clearance. These results provide compelling evidence that the developed shotgun proteomics methodology allows for the comprehensive analysis of mammalian liver membrane proteins and detailed some of the underlying changes in liver metabolism induced by the presence of a tumor. This model may reflect changes that could occur in the livers of cancer patients and has implications for drug treatments.
Table of ContentsIntroduction -- Shotgun proteomic analysis of rat liver membrane proteins -- A combination of immobilised pH gradients improve membrane proteomics -- Affects of tumor-induced inflammation on membrane proteins abundance in the mouse liver -- Affects of tumor-induced inflammation on biochemical pathways in the mouse liver -- General discussion -- References.
NotesBibliography: p. 200-212
Awarding InstitutionMacquarie University
Degree TypeThesis PhD
DegreeThesis (PhD), Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences
Department, Centre or SchoolDepartment of Chemistry and Biomolecular Sciences
Year of Award2009
Principal SupervisorMark Baker
RightsCopyright disclaimer: http://www.copyright.mq.edu.au Copyright Joel Chick 2009.
Extent609 p. ill. (some col.)
Former Identifiersmq:5970 http://hdl.handle.net/1959.14/42528 1343344
CancerLiver cells -- Molecular aspectsimmobilised pH gradientMembrane proteins -- AnalysisProteomicsisoelectric focusingMembrane proteins -- StructureMass spectrometryProteins -- Separation -- MethodologyLiver cellsProteinsProteomics -- MethodologyCancer -- Molecular aspectsMembrane proteinsIsoelectric focusingmass spectrometrymembrane proteinsshotgun proteomics