Biomarker discovery via matrix assisted laser desorption/ionization time-of-flight mass spectrometry: Tales of broccoli and breast cancer

Novel methods for the discovery of proteomic biomarkers have produced many potential candidates that could allow for early detection, risk and clinical outcome prediction for a wide range of conditions and diseases. The discovery of biomarkers in human plasma or serum that are altered with disease risk or status could offer a valuable screening tool for disease detection and management. This dissertation further develops methodology for probing deep into the "peptidome" (defined as peptides/proteins from 1,000-20,000 m/z) via Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass spectrometry (MALDI-TOF MS) to screen for plasma/serum biomarkers. Assessment of sample preparation methods allowing for consistent MALDI-TOF MS data collection are carried out. Additionally, a novel approach to immunoaffinity mass spectrometry is described wherein immobilized ligands are appended to a gold biochip surface through electrochemically cleavable linkers. While the use of this technology to detect differences in the levels of isoforms of transthyretin (TTR) revealed no significant differences in a Japanese population with and without breast cancer, the isoform ratios did differ in a significant manner when comparing control samples collected in Japan and the US. These results show the technological ability of the method to differentiate between different groups by comparing clearly defined isoforms of TTR. I also examined how the "peptidome" is altered in response to cruciferous vegetable feeding in people of different GSTM1 genotypes. Cruciferous vegetable intake, particularly in GSTM1+ individuals, caused increases and decreases in multiple proteins in human blood. Specifically, decreases in levels of TTR and ZAG were observed in human blood. The ability to differentiate populations was then applied to a time-course rat model of mammary carcinogenesis. One plausible candidate was elevated in advance of tumor detection was Dermcidin (DCD). We show that DCD levels in the case samples increase with tumor progression. In applying these findings to human samples, we also saw an increase in levels of DCD at time of breast cancer diagnosis in one sample set and in a set of pre-diagnostic breast cancer samples for non-HRT uses when compared to controls.