Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion
Abstract
An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently-attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 °C and 50 °C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 0.24 and 163.20 hours at 40 ºC, respectively. The improved stability of EC-TR can be explained by covalent-linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40 °C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of one week. In the same condition, CA-TR showed poor performance from the beginning, and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomicmore »
- Authors:
- Publication Date:
- Research Org.:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1001091
- Report Number(s):
- PNNL-SA-72385
Journal ID: ISSN 0006-3592; 32707; KP1601010; TRN: US201101%%828
- DOE Contract Number:
- AC05-76RL01830
- Resource Type:
- Journal Article
- Journal Name:
- Biotechnology and Bioengineering, 107(6):917-923
- Additional Journal Information:
- Journal Volume: 107; Journal Issue: 6; Journal ID: ISSN 0006-3592
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; AUTOLYSIS; COATINGS; DIGESTION; ENZYMES; LEACHING; PERFORMANCE; POLYMERS; PROTEINS; STABILITY; STABILIZATION; TRYPSIN; enzyme coating; trypsin; electrospun nanofibers; protein digestion; ENZYME STABILIZATION; TRYPTIC DIGESTION; IDENTIFICATION; PROTEOLYSIS; THERMOSTABILITY; IMMOBILIZATION; NANOPARTICLES; PRINCIPLES; INCREASE; Environmental Molecular Sciences Laboratory
Citation Formats
Ahn, Hye-Kyung, Kim, Byoung Chan, Jun, Seung-Hyun, Chang, Mun Seock, Lopez-Ferrer, Daniel, Smith, Richard D, Gu, Man Bock, Lee, Sang-Won, Kim, Beom S, and Kim, Jungbae. Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion. United States: N. p., 2010.
Web. doi:10.1002/bit.22895.
Ahn, Hye-Kyung, Kim, Byoung Chan, Jun, Seung-Hyun, Chang, Mun Seock, Lopez-Ferrer, Daniel, Smith, Richard D, Gu, Man Bock, Lee, Sang-Won, Kim, Beom S, & Kim, Jungbae. Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion. United States. https://doi.org/10.1002/bit.22895
Ahn, Hye-Kyung, Kim, Byoung Chan, Jun, Seung-Hyun, Chang, Mun Seock, Lopez-Ferrer, Daniel, Smith, Richard D, Gu, Man Bock, Lee, Sang-Won, Kim, Beom S, and Kim, Jungbae. 2010.
"Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion". United States. https://doi.org/10.1002/bit.22895.
@article{osti_1001091,
title = {Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion},
author = {Ahn, Hye-Kyung and Kim, Byoung Chan and Jun, Seung-Hyun and Chang, Mun Seock and Lopez-Ferrer, Daniel and Smith, Richard D and Gu, Man Bock and Lee, Sang-Won and Kim, Beom S and Kim, Jungbae},
abstractNote = {An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently-attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 °C and 50 °C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 0.24 and 163.20 hours at 40 ºC, respectively. The improved stability of EC-TR can be explained by covalent-linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40 °C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of one week. In the same condition, CA-TR showed poor performance from the beginning, and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis, but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.},
doi = {10.1002/bit.22895},
url = {https://www.osti.gov/biblio/1001091},
journal = {Biotechnology and Bioengineering, 107(6):917-923},
issn = {0006-3592},
number = 6,
volume = 107,
place = {United States},
year = {Wed Dec 15 00:00:00 EST 2010},
month = {Wed Dec 15 00:00:00 EST 2010}
}