Proteomics and beyond: Development of methodologies for early detection of cancer

Protein biomarkers have the potential to revolutionize medical diagnosis, prognosis, and treatment of cancer through early disease detection, therapy monitoring, and development of personalized medicine. The field of clinical proteomics aims at discovering disease-specific biomarkers by comparative studies of the proteomes of diseased and healthy samples. This dissertation demonstrates the significance of proteomic tools and liquid chromatography tandem mass spectrometry (LC-MS/MS) to the development of new platforms for discovery and verification/qualification of protein biomarker candidates in complex biological samples.;In order to illustrate the process of generating biomarker diagnostics (blood tests), Chapter 1 provides an overview of the biomarker development pipeline and proteomic-based technologies, which are utilized for discovery and verification of protein biomarker candidates. The advantages as well as challenges of both aspects are detailed in order to establish the basis of the relevance for developing new methodologies in this thesis. Furthermore, clear cell renal cell carcinoma (RCC) and recent reports on protein biomarker discovery in RCC are discussed.;Having established the importance of the need for new analytical methodologies and biomarkers for early detection of RCC, Chapter 2 details the application of quantitative stable isotope dimethyl labeling and two-dimensional (2-D) liquid chromatography and high mass accuracy and resolution mass spectrometry (FT-ICR MS) for comprehensive proteomic analysis of two isogenic cell lines of RCC. Subsequent extensive pathway and biological process analysis allowed for confirmation of processes activated in RCC. Key findings from this study suggest high degree of heterogeneity in the two sets of cancer samples and the differentially expressed proteins identified.;In Chapter 3 we describe the development, optimization, and validation of two different sample enrichment strategies prior to selected reaction monitoring (SRM) mass spectrometry verification of low level cell-derived biomarker candidates for RCC, carbonic anhydrase 12 (CA12) and egl nine homolog 3 (EGLN3), in complex cell lysate samples. Platform 1 (IP/Chip/SRM) consisted of antibody-based enrichment of CA12 protein on magnetic beads prior to LC-Chip SRM quantitative analysis. The alternative and novel approach, Platform 2 (Chip/Chip/SRM), utilized isoelectric focusing (IEF) on a microscale chip (digital ProteomeChip (dPC(TM))) technology for separation and enrichment of peptides derived from the target proteins. The sub-nonogram-per-milliliter sensitivity and excellent analytical performance achieved with the Chip/Chip/SRM platform led to further evaluation of the assay in plasma samples.;Therefore, Chapter 4 reports on the development and optimization of Chip/Chip/SRM platform for enrichment and quantitation of low abundance protein biomarkers in human plasma. The platform was evaluated using prostate specific antigen (PSA) as a model protein added to human female plasma samples. This study demonstrated that combination of immunodepletion of albumin and IgG along with peptide fractionation on the dPC and SRM resulted in reproducible quantitation of PSA at levels as low as 1.0 ng/mL in plasma. Furthermore, PSA levels in male prostate cancer (PCa) patient plasmas determined with the platform were well correlated with ELISA analyses of PSA. Key findings from this study suggest that the assay is a general method that can be applied to a large number of human proteins, especially targets for which antibodies are not available.;Chapter 5 presents a different perspective on early detection of cancer by use of nanotechnology for imaging of cell surface oligosaccharides and changes in glycosylation in cancer cells. A nanotechnology platform consisting of gold nanoparticles (AuNPs) functionalized with an Aleuria Aurantia Lectin (AAL), termed AAL-PEG-AuNPs, was developed and characterized. The goal of this platform was to deliver the lectin to fucose motifs presented on the cell surface and visualize the interactions by two-photon laser scanning microscopy (2PM). Qualitative differences in optical contrast between RCC cancer and healthy cells suggest that lectin-AuNPs bioconjugates have the potential for imaging differences in glycan content and/or structure in tissues or cells.