Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin, 300070, China.
Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin, 300070, China.
Anal Chim Acta. 2020 Mar 15;1102:1-10. doi: 10.1016/j.aca.2020.01.025. Epub 2020 Jan 14.
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.
基于变性和在线固定化酶反应器 (IMER) 消化的微流控平台首次在玻璃芯片上开发用于蛋白质预处理。玻璃芯片上三个入口通道和两组蛇形通道的设计可以允许蛋白质溶液、还原剂和烷化剂同时注入芯片通道，并确保反应溶液在线高效混合和充分反应。通过硫醇-烯点击化学反应，在聚（三羟甲基丙烷三甲基丙烯酸酯）整体柱的表面制备了基于毛细管和玻璃芯片的胰蛋白酶 IMER。宽流速容忍度范围（0.8-5.0 μL/min），以及可接受的重现性（RSD%=3.1%，n=5）和稳定性（2 个月内酶活性降低 13.8%）表明了 IMER 用于在线消化蛋白质的可行性。与溶液变性-离线 IMER 消化相比，芯片变性-芯片 IMER 和芯片变性-在线 IMER 的集成微流控平台对于具有相似数量蛋白质（798 或 826 与 843）和独特肽（3923 或 4593 与 3916）的小鼠肝蛋白具有相当的蛋白质鉴定能力。更重要的是，蛋白质样品的快速消化和与 MS 的可能结合表明，这种微流控平台可以成为快速蛋白质组学分析的潜在方法。
