Rapid protein digestion and identification using monolithic enzymatic microreactor coupled with nano-liquid chromatography-electro spray ionization mass spectrometry

被引：76

作者：

Duan, JC ^{[1
]}

Sun, LL ^{[1
]}

Liang, Z ^{[1
]}

Zhang, J ^{[1
]}

Wang, H ^{[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：

JOURNAL OF CHROMATOGRAPHY A | 2006年 / 1106卷 / 1-2期

关键词：

monolithic column; enzymatic reactor; protein identification; nano-liquid chromatography-tandem mass spectrometry;

D O I：

10.1016/j.chroma.2005.11.102

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A novel monolithic enzymatic microreactor was prepared in the fused-silica capillary by in situ polymerization of acrylamide (AA), Nacryloxysuccinimide (NAS) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, which could offer very low back pressure, enabling the fast digestion of proteins. The performance of the monolithic microreactor was demonstrated by digesting cytochrome c at high flow rate, and the comparisons between the in-solution digestion and on-column reaction were made by a nano-high performance liquid chromatography-mass spectrometry (nano-fIPLC-MS) system. The performance of the monolithic microreactor was demonostrated with the digestion of cytochrome c at the fast flow rate of 1 mu L-min, which afforded a residence time of 7 s, yielding a sequence coverage of 54.81% using strict multiple database searching thresholds. Furture more, a mixture of four standard proteins was digested and analyzed using the on-line digestion and nano-HPLC-MS system. The results showed the promising of such a system in the analysis of protein mixture. (c) 2005 Elsevier B.V. All rights reserved.

引用

页码：165 / 174

页数：10