William S. Hancock
Paul Vouros, David A. Forsyth, Zhaohui S. Zhou, Maureen Hattersley
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Science. Department of Chemistry and Chemical Biology.
Breast Cancer, LC/MS, MCF-7, M-LAC, Mouse, Proteomic
Breast - Diseases - Diagnosis, Clinical medicine - Computer programs, Proteomics
This thesis is based in the area of clinical proteomics and relies primarily on LC/MS analytical methods to search for disease associated changes in blood. Computer programming was investigated as a solution to issues of data management and interpretation.
Chapter 1 introduces the analytical methods employed in this thesis including HPLC, mass spectrometry and protein fractionation methods. Background information regarding the disease processes of breast cancer and metabolomic disorders is also provided.
Chapter 2 describes a mouse plasma proteomic analysis aimed to uncover protein biomarkers of the growth of human breast cancer cells implanted in immunocompromised mice. M-LAC fractionation removed the serum albumin and enriched the glycoproteins prior to tryptic digestion and LC/MS analysis. Several proteins were observed to have significant changes in abundance including changes in the abundance of EGFR. Samples analyzed using the LTQ-FT provided high mass accuracy data which was used to discover tumor specific proteins with peptide sequences of human origin.
Chapter 3 describes testing of serum from gastric bypass patients, who developed hyperinsulinemia, through the use of multiple testing methods in hopes of finding the origin of the disease. Two types of abundant protein depletion, 12-protein depletion and 2-protein depletion, were used in separate experiments prior to M-LAC fractionation, tryptic digestion and LC-MS analysis for one part of the study. Of the possible proteins involved in the disease symptoms Vitamin D Binding protein (VDBP) appears as particularly important. In the next experiment the peptidome of the serum samples was investigated to find endogenous peptides which might play a role in hyperinsulinemia. Apolipoproteins A-1, A-IV and C-III all show reduced endogenous enzymatic cleavage and are known to be associated with lipid transport and type 2 diabetes. A third experiment investigated the possibility of an autoimmune cause for the complications through the use of Western Blotting. Preliminary results from the experiment indicate that there is an autoimmune response or cause of hyperinsulinaemia in gastric bypass patients.
Chapter 4 deals with interpretation of the data produced by clinical proteomics experiments through the use of specialized computer programs. ProteinCenter speeds up identifying the biological roles of the proteins discovered during the analysis. We also explore the possibility of utilizing the LC/MS data from proteomic experiments without identifying proteins to discover disease or health patterns in the chromatographic output.
Chapter 5 discusses what improvements are imminent in the field of clinical proteomics in regards to sample quality and processing time. In the immediate future computer programming will play a significant role in the process of analyzing LCMS data.
Orazine, Christina, "Approaches towards clinical proteomic studies in blood" (2010). Chemistry Dissertations. Paper 41. http://hdl.handle.net/2047/d20002799
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