National Chromatographic R. & A. Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, China.
Anal Chem. 2010 Mar 15;82(6):2574-9. doi: 10.1021/ac902835p.
An integrated sample treatment device, composed of a membrane interface and a monolithic hybrid silica based immobilized enzymatic reactor (IMER), was developed for the simultaneous sample buffer exchange, protein enrichment, and online digestion, by which for the sample buffer, the acetonitrile content was reduced to approximately 1/10 of the initial one, and the pH value was adjusted from approximately 3.0 to approximately 8.0, compatible for online trypsin digestion. Furthermore, the signal intensity of myoglobin digests was improved by over 10 times. Such an integrated device was successfully applied to the online treatment of three protein eluates obtained by reverse-phase liquid chromatography (RPLC) separation, followed by further protein digest analysis with microreverse-phase liquid chromatography-electrospray ionization-tandem mass spectrometry (microRPLC-ESI-MS/MS). The experimental results showed that the performance of such an integrated sample treatment device was comparable to that of the traditional offline sample treatment method, including lyophilization and in-solution digestion. However, the consumed time was reduced to 1/192. All these results demonstrate that such an integrated sample treatment device could be further online coupled with protein separation, peptide separation, and identification, to achieve high-throughput proteome analysis.
一种集成的样品处理装置，由膜接口和整体式混合硅胶固定化酶反应器（IMER）组成，用于同时进行样品缓冲液交换、蛋白质富集和在线消化，通过该装置，样品缓冲液中的乙腈含量降低至初始值的约 1/10，pH 值从约 3.0 调节至约 8.0，适用于在线胰蛋白酶消化。此外，肌红蛋白消化物的信号强度提高了 10 多倍。该集成装置成功应用于反相液相色谱（RPLC）分离得到的三种蛋白质洗脱液的在线处理，然后通过微反相液相色谱-电喷雾电离-串联质谱（microRPLC-ESI-MS/MS）进一步进行蛋白质消化分析。实验结果表明，这种集成的样品处理装置的性能与传统的离线样品处理方法（包括冻干和溶液中消化）相当。然而，所消耗的时间减少到 1/192。所有这些结果表明，这种集成的样品处理装置可以进一步与蛋白质分离、肽分离和鉴定在线耦合，以实现高通量蛋白质组分析。
