Rapid protein identification using monolithic enzymatic microreactor and LC-ESI-MS/MS

被引：83

作者：

Duan, JC ^{[1
]}

Liang, Z ^{[1
]}

Yang, C ^{[1
]}

Zhang, J ^{[1
]}

Zhang, LH ^{[1
]}

Zhang, WB ^{[1
]}

Zhang, YK ^{[1
]}

机构：

[1] Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog Res & Anal Ctr, Dalian 116023, Peoples R China

来源：

PROTEOMICS | 2006年 / 6卷 / 02期

关键词：

enzymatic reactor; LC-MS/MS; monolithic column; protein identification;

D O I：

10.1002/pmic.200500234

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. in addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.

引用

页码：412 / 419

页数：8

共 37 条

[1] Improved performance in silicon enzyme microreactors obtained by homogeneous porous silicon carrier matrix [J].