- Peptide mapping using capillary electrophoresis (CE)
- Immobilized Enzyme Reactor (IMER)
- Crosslinking enzyme using glutaraldehyde
Due to their selectivity, enzymes have been used in analytical chemistry for a wide range of assays. Enzyme immobilization is a novel method whereby autoproteolysis decreases significantly; therefore, a proteolytic enzyme can be used at higher enzyme-to-substrate ratio for faster reaction. Immobilized enzyme reactors, once seen only in process chemistry, are being used more frequently in proteomics studies for protein digestion.
The analytical performance of an immobilized enzyme microreactor (IMER) is directly affected by the immobilization process. My research aims to develop innovative strategies for high-resolution separations and quantitative detection of biomolecules. The significance of the study includes identifying protein biomarkers by providing high enough sensitivity to determine whether a protein or a metabolite in a biological sample is at normal range, or over- or under-produced due to disease and/or therapy.
The immobilization strategy of using GA as an inexpensive and commercially available protein crosslinking agent avoids the need for a solid support. Ideally, a porous network of GA-enzyme across the capillary would provide fast mass transfer from the reduced diffusion path length of substrate and, thus, increase the enzyme contact time.
This project provides opportunities in which students will become familiar with the enzyme immobilization procedure, the concept of autoproteolysis, digestion, and denaturation. They will participate in optimizing the immobilization conditions both in batch and IMER format by varying the experimental parameters one step at the time and making conclusions to choose the optimum conditions based on their results. Students will receive training on several instruments including CE, HPLC and MS systems and learn how to operate and program these instruments independently.