Premium
Rapid protein identification using monolithic enzymatic microreactor and LC‐ESI‐MS/MS
Author(s) -
Duan Jicheng,
Liang Zhen,
Yang Chun,
Zhang Jie,
Zhang Lihua,
Zhang Weibing,
Zhang Yukui
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200500234
Subject(s) - chemistry , chromatography , microreactor , glutaraldehyde , trypsin , aqueous solution , polymerization , acrylamide , monolithic hplc column , monomer , high performance liquid chromatography , enzyme , organic chemistry , polymer , catalysis
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.