- Signal transduction within and between cells is at the core of biological activity in all living systems. Signaling networks are required for regulating biological functions, including growth, development and survival. Deregulation of signaling cascades has been linked to chronic and acute diseases and disorders This thesis focuses on mass spectrometry as a high resolution and high mass accuracy technique for the detection and characterization of proteins in biological systems.
The thesis presents applications of contemporary mass-spectrometric methods to identify proteins, determine changes in their expression levels, and characterize post-translational modifications, in an effort to study changes in cell signaling in response to stress or disease. Various sample preparation methods to successfully suit needs of different biological questions were developed and applied: (1) extraction of the proteome, (2) chemical tagging and enrichment of the ATPome, a sub proteome comprising nucleotide binding proteins in particularly ATP-binding proteins including kinases, and (3) metal affinity complexation and enrichment of phosphopeptides. We used the following hybrid mass analyzer configurations: a quadrupole time-of-flight (qToF) instrument with ion mobility, a linear ion trap hyphenated with a FT-ICR mass spectrometry (LTQ-FT) and a hybrid ion trap-orbitrap mass spectrometer (Orbitrap Elite). The bioinformatic analysis of the proteomics data required multiple combinations of software packages to sequence proteins, perform absolute and relative quantification, statistically analyze and visualize data.
Chapter 3 describes the use of zebrafish (Danio rerio) as one of the few vertebrate models that similar to humans cannot synthesize vitamin C to investigate the system-wide consequences of deficiencies in two essential micronutrients, vitamins E and C, on the proteome biology. A label-free proteomics workflow was applied to detect changes in protein abundance estimates dependent on vitamin regimes. The study reveals suppression in an energy metabolism cycle, glycolysis, in vitamin C and E deficient zebrafish. It was discovered that alternative energy cycle, glutaminolysis, is activated to fulfill energy requirement.
Chapter 4 focuses on the determination of proteome differences that can be linked to the propensity of metastasis in osteosarcoma (OS), a bone cancer that predominantly targets the adolescent age group. OS has a high propensity to metastasize to the lungs, which is associated with a poor prognosis. The study utilizes canine osteosarcoma cell lines that were originally obtained from orthotropic primary OS and metastatic cells. Canis familiaris, the domestic dog, is an established large animal model of OS that recapitulate
many biological and clinical features of the human malignancy. We applied a two-prone comparative proteomics approach that consisted of: (a) determination of protein abundance levels and (b) focus on kinases, a functional sub-proteome, using a chemical affinity tag for enrichment of ATP-binding proteins. Findings of this study indicated that in the highly metastasizing canine osteosarcoma cell line proteins associated with extracellular adhesion were deregulated, which may enhance metastogenesis. Mitogen activated protein kinases MAP2K6, MAP4K3, MAP4K4, MAP4K5, ZAK and v-akt murine thymoma viral oncogene homolog 1, AKT1, are among those expressed in significantly lower abundance in the highly metastasizing canine osteosarcoma cell line indicating changes in cell signaling.
Chapter 5 describes the development and application of a multiple protease protocol (Trypsin, LysC, AspN, Chymotrypsin and GluC) for improving the number of phosphosite identifications in a large-scale phosphoproteomics studies. The method combines immobilized titanium ion affinity chromatography (Ti⁴⁺-IMAC) with a data-dependent, decision tree-based data acquisition technique utilizing two complementary fragmentation methods, namely collision induced dissociation (CID) and electron transfer dissociation. The multiple protease protocol was applied to human leukemic T cell lymphoblasts (Jurkat E6.1) and resulted in the detection of >11,000 unique phosphosites, the most comprehensive identification among methods that use similar phosphopeptide enrichment approach.