Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity

Junfeng Ma, Zhen Liang, Xiaoqiang Qiao, Qiliang Deng, Dingyin Tao, Lihua Zhang, Yukui Zhang

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2949-2956
Number of pages8
JournalAnalytical Chemistry
Volume80
Issue number8
DOIs
StatePublished - Apr 15 2008
Externally publishedYes

Fingerprint

Silicon Dioxide
Trypsin
Myoglobin
Liquid chromatography
Glutaral
Escherichia coli
Sol-gel process
Mass spectrometry
Proteins
Polymerization
Monitoring

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity. / Ma, Junfeng; Liang, Zhen; Qiao, Xiaoqiang; Deng, Qiliang; Tao, Dingyin; Zhang, Lihua; Zhang, Yukui.

In: Analytical Chemistry, Vol. 80, No. 8, 15.04.2008, p. 2949-2956.

Research output: Contribution to journalArticle

Ma, J, Liang, Z, Qiao, X, Deng, Q, Tao, D, Zhang, L & Zhang, Y 2008, 'Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity', Analytical Chemistry, vol. 80, no. 8, pp. 2949-2956. https://doi.org/10.1021/ac702343a
Ma, Junfeng ; Liang, Zhen ; Qiao, Xiaoqiang ; Deng, Qiliang ; Tao, Dingyin ; Zhang, Lihua ; Zhang, Yukui. / Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity. In: Analytical Chemistry. 2008 ; Vol. 80, No. 8. pp. 2949-2956.
@article{77f2d5cff7b04bb7ac060fab1dd62248,
title = "Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity",
abstract = "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.",
author = "Junfeng Ma and Zhen Liang and Xiaoqiang Qiao and Qiliang Deng and Dingyin Tao and Lihua Zhang and Yukui Zhang",
year = "2008",
month = "4",
day = "15",
doi = "10.1021/ac702343a",
language = "English (US)",
volume = "80",
pages = "2949--2956",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "8",

}

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.

UR - http://www.scopus.com/inward/record.url?scp=42349103426&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=42349103426&partnerID=8YFLogxK

U2 - 10.1021/ac702343a

DO - 10.1021/ac702343a

M3 - Article

C2 - 18333626

AN - SCOPUS:42349103426

VL - 80

SP - 2949

EP - 2956

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 8

ER -