Integrated microfluidic chip for on-line proteome analysis with combination of denaturing and rapid digestion of protein.
A microfluidic platform based on the integration of denaturation and online immobilized enzyme reactor (IMER) digestion for protein pretreatment was first developed on a glass chip. The design of three inlet channels and two groups of snake channel in glass chip can allow the protein solution, the reducing reagent and the alkylating agent to be simultaneously injected into the chip channel and ensured the reaction solution on-line efficient mixing and sufficient reacting. By thiol-ene click chemistry, the capillary-based and glass chip-based trypsin IMER on the surface of poly(trimethylolpropane trimethacrylate) monolith were fabricated. The wide range of flow rate tolerance (0.8-5.0 μL/min), and the acceptable reproducibility (RSD% = 3.1%, n = 5) and stability (13.8% decrease of enzyme activity in 2 months) indicated the feasibility of using IMER for online digestion of proteins. Compared with the solution denaturation-offline IMER digestion, the integrated microfluidic platform of chip denaturation-chip IMER and chip denaturation-online IMER have comparable protein identification ability for mouse liver protein with a similar number of protein (798 or 826 vs. 843) and unique peptides (3923 or 4593 vs. 3916). More importantly, the easy and fast digestion of protein samples and possible combination with MS revealed that this microfluidic platform can be a potential method for rapid proteomics analysis.
['Wei|Zehui|Z|', 'Fan|Peiru|P|', 'Jiao|Yajie|Y|', 'Wang|Yang|Y|', 'Huang|Yanping|Y|', 'Liu|Zhaosheng|Z|']
D000818:Animals D002417:Cattle D004229:Dithiothreitol D004800:Enzymes, Immobilized D007460:Iodoacetamide D056656:Lab-On-A-Chip Devices D008099:Liver D051379:Mice D046210:Microfluidic Analytical Techniques D011489:Protein Denaturation D059748:Proteolysis D020543:Proteome D040901:Proteomics D012710:Serum Albumin, Bovine D014357:Trypsin
Microfluidic chip Proteomics Immobilized enzyme reactor Online digestion Mouse liver protein Online degeneration