Faculty

Ph.D. Chemistry, Université de Montréal, Montreal, QC

Title of Thesis: Characterization of Glutaraldehyde-Immobilized Chymotrypsin and an In-Situ Immobilized Enzyme Reactor Using Capillary Electrophoresis-Based Peptide Mapping.

Advisor: Professor Karen C. Waldron

M.Sc. Analytical Chemistry, K. N. Toosi University of Technology, Tehran, Iran

Title of Thesis: Design and Construction of Optodes for Simultaneous Detection of Gallium, Nickel and Indium Ions, on Neutral Ionophores and Indicator Dyes

Advisor: Professor Ali Jabbari

B.Sc. Chemistry, Azad University, Tehran, Iran

Proteins are central to human physiology and disease, making protein biomarkers essential for clinical diagnosis and treatment monitoring. To analyze complex proteomes, bottom-up proteomics serves as the standard methodology by utilizing enzymes to digest large proteins into smaller, more manageable peptides for accurate mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. However, traditional in-solution digestion suffers from low throughput, requiring low enzyme concentrations and overnight incubation to avoid autolytic self-digestion, while also preventing enzyme reuse and increasing costs.

To overcome these throughput and cost limitations, our lab has developed a novel method to entrap proteolytic enzymes—such as pepsin, trypsin, and chymotrypsin—within a hydrogel matrix formed via alginate polymerization.

This ultra-rapid, simple approach eliminates enzyme autolysis and slashes digestion times from hours down to minutes while enabling enzyme reusability. Beyond drastically accelerating sample turnover, the simplicity of this hydrogel system makes it uniquely suited for automation and field-deployable, on-site protein analysis, promising broader accessibility across medicine and environmental science.

Selected Publications

• Arad M., Frey C., Balagtas R., Hare R., Ku K., Jereb D., Nestman Z., Sidhu A., Shi Y., Fordwour O., Moon K., Foster L., Ghafourifar G. (2024) Development of an Automated, Ultra-Rapid Bottom-Up Proteomics Workflow Utilizing Alginate-Based Hydrogels, Analytical Chemistry, 96, 18880-18889
• Arad M., Ku K., Frey C., Hare R., McAfee A., Ghafourifar G., Foster L. J. (2024) What proteomics has taught us about honey bee (Apis mellifera) health and disease. Proteomics, 2400075.
• Frey C., Arad M., Ky K., Hare R., Balagtas R., Shi Y., Moon K., Foster L., Ghafourifar G. (2024) Development of automated proteomic workflows utilizing silicon-based coupling agents. Journal of Proteomics, 303, 105215.
• Ku K., Frey C., Arad M., Ghafourifar G. (2022) Development of a Novel Enzyme Immobilization Method Employing Formaldehyde and TESB To Fabricate Immobilized Enzyme Microreactor for Bottom-Up Proteomics. Analytical Methods, 14, 4053-4063.
• Ghafourifar G., Waldron K.C. (2015) Fluorescence microscopy imaging of an immobilized enzyme microreactor to investigate glutaraldehyde-mediated crosslinking of chymotrypsin. Analytical Letters; 49 (4), 589-600
• Ghafourifar G., Waldron K.C. (2015) A Study of the Chymotrypsin IMER Digestion Efficiency as a Function of Substrate Size. Analytical Sciences (Revised version submitted)
• Ghafourifar G., Waldron K.C. (2015) Capillary Electrophoretic Peptide Mapping to Probe the Immobilization/Digestion Conditions of Glutaraldehyde-crosslinked Chymotrypsin. Current Analytical Chemistry; 12 (1), 65-73
• Ghafourifar G., Fleitz A., Waldron K.C. (2013) Development of glutaraldehyde-crosslinked chymotrypsin and an in situ immobilized enzyme reactor with peptide mapping by capillary electrophoresis. Electrophoresis; 34, 1804–1811
• Ghasemi J., Ahmadi-Golsefidi M., Jalilian H, Ghafourifar G., Rajabzadeh R. (2007) Simultaneous spectrophotometric determination of trace amounts of gallium and nickel by benzo 15-crown-5 And PAN using orthogonal signal correction partial least squares. The Analytical Chemistry; 3 (4-6), 1-8