Ultrafast enzymatic digestion of proteins by microdroplet mass spectrometry
Ultrafast enzymatic digestion of proteins by microdroplet mass spectrometry
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DOI：
10.1038/s41467-020-14877-x
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发表时间：
2020-02-26
影响因子：
16.6
通讯作者：
Zare, Richard N.
中科院分区：
文献类型：
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作者：
Zhong, Xiaoqin;Chen, Hao;Zare, Richard N.
用于蛋白质测序的酶消化通常需要很多时间，并且并不总是导致高序列覆盖率。在这里，我们报告了使用水性微滴来加速酶促反应，特别是改善蛋白质测序。当含有10 μ M肌红蛋白和5 μ gmL（-1）胰蛋白酶的室温水溶液从自制装置中超声喷射（-3 kV）以产生微小的（类似于9 μ m）微滴，我们在不到1 ms的消化时间内获得100%的序列覆盖率，与通过在37 ℃下孵育相同溶液14小时，然后用产生较大（类似于60 μ m）液滴的商业电喷雾电离源进行分析所获得的60%覆盖率形成鲜明对比。我们还确认了治疗性抗体曲妥珠单抗（类似于148 kDa）的序列，轻链的序列覆盖率为100%，重链的序列覆盖率为85%，证明了微滴在药物开发中的实用性。
Enzymatic digestion for protein sequencing usually requires much time, and does not always result in high sequence coverage. Here we report the use of aqueous microdroplets to accelerate enzymatic reactions and, in particular, to improve protein sequencing. When a room temperature aqueous solution containing 10 mu M myoglobin and 5 mu gmL(-1) trypsin is electrosonically sprayed (-3 kV) from a homemade setup to produce tiny (similar to 9 mu m) microdroplets, we obtain 100% sequence coverage in less than 1ms of digestion time, in sharp contrast to 60% coverage achieved by incubating the same solution at 37 degrees C for 14 h followed by analysis with a commercial electrospray ionization source that produces larger (similar to 60 mu m) droplets. We also confirm the sequence of the therapeutic antibody trastuzumab (similar to 148 kDa), with a sequence coverage of 100% for light chains and 85% for heavy chains, demonstrating the practical utility of microdroplets in drug development.