Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS
Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS
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DOI：
10.1016/j.jasms.2008.03.015
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发表时间：
2008-07-01
影响因子：
3.2
通讯作者：
Murray, Kermit K.
中科院分区：
文献类型：
--
作者：
Lee, Jeonghoon;Musyimi, Harrison K.;Murray, Kermit K.
构建了一个自动化的蛋白水解消化生物反应器和液滴沉积系统，该系统具有塑料微流控装置，用于与基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）离线接口。微流控芯片在聚（甲基丙烯酸甲酯）（PMMA）中制造，使用微研磨机并结合生物反应器，其为100 μ m宽，100 μ m深，并具有4cm的有效通道长度（400 nL体积）。芯片通过压力驱动流操作，并安装在机器人级分收集器系统上。PMMA生物反应器含有表面固定的胰蛋白酶，其使用偶联试剂N-（3-二甲基氨基丙基）-N '-乙基碳二亚胺盐酸盐（EDQ）和羟基磺基琥珀酰亚胺（磺基-NHS）共价连接到UV改性的PMMA表面。将消化的肽与芯片上的MALDI基质混合，并作为离散点沉积在MALDI靶标上。生物反应器以1 μ L/min的流速提供测试蛋白质细胞色素c的有效消化，产生约24秒的反应时间，以给出用于蛋白质鉴定的足够序列覆盖。还使用该固相生物反应器评价了其他蛋白质。通过监测细胞色素c、牛血清白蛋白（BSA）、肌红蛋白和磷酸化酶B的序列覆盖率分别为64%、35%、58%和47%，评价了消化效率。
An automated proteolytic digestion bioreactor and droplet deposition system was constructed with a plastic microfluidic device for off-line interfacing to matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microfluidic chips were fabricated in poly(methyl methacrylate) (PMMA), using a micromilling machine and incorporated a bioreactor, which was 100 ttm wide, 100 /im deep, and possessed a 4 cm effective channel length (400 nL volume). The chip was operated by pressure-driven flow and mounted on a robotic fraction collector system. The PMMA bioreactor contained surface immobilized trypsin, which was covalently attached to the UV-modified PMMA surface using coupling reagents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDQ and hydroxysulfosuccinimide (sulfo-NHS). The digested peptides were mixed with a MALDI matrix on-chip and deposited as discrete spots on MALDI targets. The bioreactor provided efficient digestion of a test protein, cytochrome c, at a flow rate of I pL/min, producing a reaction time of -24 s to give adequate sequence coverage for protein identification. Other proteins were also evaluated using this solid-phase bioreactor. The efficiency of digestion was evaluated by monitoring the sequence coverage, which was 64%, 35%, 58%, and 47% for cytochrome c, bovine serum albumin (BSA), myoglobin, and phosphorylase b, respectively.