Date of Award
Doctor of Philosophy (PhD)
Stephen J. Beebe
O. John Semmes
Time-of-flight mass spectrometry continues to evolve as a promising technique for serum protein expression profiling and biomarker discovery. As seen in our initial SELDI-TOF MS and MALDI-TOF MS profiling study of serum for the assessment of breast cancer risk, many profiling strategies typically employ single chemical affinity beads or surfaces to decrease sample complexity of dynamic fluids like serum. However, most proteins, captured on a particular surface or bead, are not resolved in the lower mass range where mass spectrometers are most effective. To this end we have designed an expression profiling workflow that utilizes immobilized trypsin paramagnetic beads in order to reduce large mass proteins into peptides that are in the ideal mass range for serum expression profiling as well as for direct LIFT-MS/MS sequence determinations. We demonstrate that this bead-based trypsinization is efficient in digesting large serum proteins in short incubation times and is highly reproducible and amenable to an automated platform. Additionally, we show that this workflow may be combined in tandem with many different types of bead fractionation surfaces. Furthermore, by utilizing two different pooled human serum sample cohorts as proof-of-concept experiments, we are able to demonstrate the reproducibility of this method in the profiling of clinical samples and the ease of differential peptide identity determination. Overall, this method is an attractive strategy for high-throughput serum profiling with the goal of detecting and identifying differential peptides.
Karbassi, Izabela D..
"The Application of Innovative High-Throughput Techniques to Serum Biomarker Discovery"
(2008). Doctor of Philosophy (PhD), Dissertation, Biological Sciences, Old Dominion University, DOI: 10.25777/a00s-qf63