Membrane-based nanoscale proteolytic reactor enabling protein digestion, peptide separation, and protein identification using mass spectrometry
Membrane-based nanoscale proteolytic reactor enabling protein digestion, peptide separation, and protein identification using mass spectrometry
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DOI：
10.1021/ac025768b
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发表时间：
2003-03-01
影响因子：
7.4
通讯作者：
Lee, CS
中科院分区：
文献类型：
--
作者：
Cooper, JW;Chen, JZ;Lee, CS
一个小型化的胰蛋白酶膜反应器内的常用的毛细管配件的开发和证明，使快速和灵敏的蛋白质鉴定，通过在线蛋白水解消化和分析的蛋白酶使用纳米-ESI-MS和MALDI-MS。的设计和组装的毛细管配件为基础的胰蛋白酶膜反应器是简单和高度稳健的，而不需要昂贵的制造技术和程序。所得的蛋白质电泳物还可以在集成平台中的质谱分析之前使用毛细管反相液相色谱或瞬时毛细管等速电泳/区带电泳进一步浓缩和解析。通过将这些结果与我们先前使用塑料微流体的研究获得的结果进行比较（Gao等人，AnaL Chem.2001，73，2648-2655），使用这种新开发的胰蛋白酶消化站可以实现死体积和样品消耗的显著减少。这种纳米级反应系统能够在几秒钟而不是几小时内快速蛋白水解消化小于10（-8）M的蛋白质浓度，消耗非常少的样品（小于或等于5 fmol），并提供与各种分离和质谱技术的毛细管接口。用于在蛋白质鉴定中实现完全肽覆盖的超快酶促周转是由膜介质的高度多孔结构贡献的，其提供过量的胰蛋白酶加载，同时消除扩散限制反应动力学的约束。
A miniaturized trypsin membrane reactor housed inside a commonly used capillary fitting is developed and demonstrated for enabling rapid and sensitive protein identification by on-line proteolytic digestion and analysis of protein digests using nano-ESI-MS and MALDI-MS. The design and assembly of the capillary fitting-based trypsin membrane reactor are straightforward and highly robust, without the need for expensive fabrication technology and procedures. The resultant protein digests can also be further concentrated and resolved using capillary reversed-phase liquid chromatography or transient capillary isotachophoresis/zone electrophoresis prior to the mass spectrometric analysis in an integrated platform. By comparing these results with the results obtained from our previous studies using plastic microfluidics (Gao et al., AnaL Chem. 2001, 73, 2648-2655), significant reduction in dead volume and sample consumption can be achieved using this newly developed tryptic digestion station. This nanoscale reaction system enables rapid proteolytic digestion in seconds instead of hours for a protein concentration of less than 10(-8) M, consumes very little sample (less than or equal to 5 fmol), and offers capillary interfaces with various separation and mass spectrometry techniques. The ultrafast enzymatic turnover for attaining complete peptide coverage in protein identification is contributed by the highly porous structure of the membrane media, providing excessive trypsin loading while eliminating the constraints of diffusion-limited reaction kinetics.