Technologies for Proteomic and Genomic Biomarker Analysis

In the first part of this dissertation, we systematically validated the application of molecular weight cut-off ultrafiltration in separation and enrichment of low-molecular-weight peptides from human serum. Under optimized conditions, both free-phase and bound LMW peptides could be separated and enriched. The method proved to be highly efficient and reproducible coupled with MALDI-TOF MS proteomic pattern analysis. Three marker peaks were found to be eligible for distinguishing normal and ovarian cancer samples. A novel organic solvent precipitation method coupled with enzymatic deglycosylation was also developed for biomarker detection from human serum. This method allowed us to generate reproducible free-phase peptide patterns comparing with the ultrafiltration method. A potential marker was found up-regulated in benign and ovarian cancer patients. It was further identified as des-alanine-fibrinopeptide A using LC tandem mass spectrometry. In the second part of this dissertation, a new sample preparation procedure was developed to improve the MALDI-TOF analysis of low-concentration oligonucleotides. The oligonucleotide solutions are first dispensed and allow shrinking onto a small spot on an anchoring target. A small volume (0.1uL) of saturated 3-HPA matrix solution is then added on top of each dried oligonucleotide spot. Samples prepared by this procedure are homogenous and reduces the need to search for \u27sweet\u27 spots. The increased shot-to-shot and sample-to-sample reproducibility makes it useful for high-throughput quantitative analysis. This procedure allowed robust detection of oligonucleotides at 0.01℗æM level and mini-sequencing products produced using only 50 fmol of extension primer. And a strategy called probe-clamping-primer-extension-PCR (PCPE-PCR) was developed to detect MRS alterations in a large background of wild-type DNA. PCR errors often generate false positive mutant alleles. In PCPE-PCR, mutant single-strand DNA molecules are preferentially produced and enriched. Thereafter, the r