TY - JOUR
T1 - Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity
AU - Ma, Junfeng
AU - Liang, Zhen
AU - Qiao, Xiaoqiang
AU - Deng, Qiliang
AU - Tao, Dingyin
AU - Zhang, Lihua
AU - Zhang, Yukui
PY - 2008/4/15
Y1 - 2008/4/15
N2 - A novel kind of immobilized trypsin reactor based on organic-inorganic hybrid silica monoliths has been developed. With the presence of cetyltrimethyl ammonium bromide (CTAB) in the polymerization mixture, the hybrid silica monolithic support was prepared in a 100 μm i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, the monolith was activated by glutaraldehyde, and trypsin was covalently immobilized. By monitoring the reaction of a decapeptide, C-myc (EQKLISEEDL), the enzymatic activity of the immobilized trypsin was calculated, and the results showed that the digestion speed was about 6600 times faster than that performed in free solution. The performance of such a microreactor was further demonstrated by digesting myoglobin, with the digested products analyzed by microflow reversed-phase liquid chromatography coupled with tandem mass spectrometry (μRPLC-MS/MS). With a stringent threshold for the unambiguous identification of the digests, the yielding sequence coverage for on-column digestion was 92%, the same as that obtained by insolution digestion, whereas the residence time of myoglobin in the former case was only 30 s, about 1/1440 of that performed in the latter case (12 h). Moreover, such an immobilized trypsin reactor was also successfully applied to the digestion of a mixture of model proteins and proteins extracted from E. coli.
AB - A novel kind of immobilized trypsin reactor based on organic-inorganic hybrid silica monoliths has been developed. With the presence of cetyltrimethyl ammonium bromide (CTAB) in the polymerization mixture, the hybrid silica monolithic support was prepared in a 100 μm i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, the monolith was activated by glutaraldehyde, and trypsin was covalently immobilized. By monitoring the reaction of a decapeptide, C-myc (EQKLISEEDL), the enzymatic activity of the immobilized trypsin was calculated, and the results showed that the digestion speed was about 6600 times faster than that performed in free solution. The performance of such a microreactor was further demonstrated by digesting myoglobin, with the digested products analyzed by microflow reversed-phase liquid chromatography coupled with tandem mass spectrometry (μRPLC-MS/MS). With a stringent threshold for the unambiguous identification of the digests, the yielding sequence coverage for on-column digestion was 92%, the same as that obtained by insolution digestion, whereas the residence time of myoglobin in the former case was only 30 s, about 1/1440 of that performed in the latter case (12 h). Moreover, such an immobilized trypsin reactor was also successfully applied to the digestion of a mixture of model proteins and proteins extracted from E. coli.
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U2 - 10.1021/ac702343a
DO - 10.1021/ac702343a
M3 - Article
C2 - 18333626
AN - SCOPUS:42349103426
SN - 0003-2700
VL - 80
SP - 2949
EP - 2956
JO - Analytical chemistry
JF - Analytical chemistry
IS - 8
ER -