Wed, 28 Sep 2022
Events, Seminar Series

Dr. Karen Waldron

Université de Montréal

Crosslinked proteolytic enzymes for microscale proteomics applications

Wednesday, September 28, 2022
SSB 7172 @ 3:30 p.m.

Host: Dr. Bingyun Sun



Proteolytic enzymes are routinely used to cleave the potentially thousands of proteins in a biological sample into smaller, more manageable peptide fragments. This facilitates their separation and identification by nano-HPLC, capillary electrophoresis (CE) and/or mass spectrometry (MS). This process, called peptide mapping, is one of the steps used in the workflow for identifying, quantifying, characterizing or probing the function of proteins in the vast field of proteomics, specifically “bottom-up” proteomics. Insoluble proteolytic enzymes (i.e., trypsin, chymotrypsin, pepsin, Lys-C, Asp-N, etc.) offer several benefits such as limited autolysis, reusability and rapid digestion because high enzyme-to-substrate ratios can be used. The added advantage of adaptability to microreactor formats and microfluidic platforms comes from using glutaraldehyde (GA) to crosslink enzymes and render them insoluble instead of immobilizing them on solid-phase supports. In 2004 we reported a multi-step GA-crosslinking procedure for trypsin and compared its specific activity and peptide maps to trypsin immobilized onto GA-functionalized glass beads. The procedure was later adapted for chymotrypsin to digest nanomolar concentrations of fluorescently labelled protein substrate with peptide mapping by CE laser-induced fluorescence (LIF) detection. A GA-chymotrypsin microreactor was fabricated in a capillary column by programing reagent delivery in a CE instrument. Despite the ease in making GA-crosslinked trypsin and chymotrypsin “particles”, their re-use for multiple digestions has been problematic. Our successes and setbacks will be discussed, as well as our efforts to improve the robustness of the crosslinked enzymes using a Design-of-Experiments (DOE) approach to investigate the experimental parameters in our multi-step crosslinking method.