Efficient protein digestion using highly-stable and reproducible trypsin coatings on magnetic nanofibers

Byoungsoo Lee; Byoung Chan Kim; Mun Seock Chang; Han Sol Kim; Hyon Bin Na; Yong Il Park; Jinwoo Lee; Taeghwan Hyeon; Hookeun Lee; Sang Won Lee; Jungbae Kim

doi:10.1016/j.cej.2015.12.003

Efficient protein digestion using highly-stable and reproducible trypsin coatings on magnetic nanofibers

Byoungsoo Lee, Byoung Chan Kim, Mun Seock Chang, Han Sol Kim, Hyon Bin Na, Yong Il Park, Jinwoo Lee, Taeghwan Hyeon, Hookeun Lee, Sang Won Lee, Jungbae Kim

Research output: Contribution to journal › Article › peer-review

14 Citations (Scopus)

Abstract

Protein digestion, using an enzyme called trypsin (TR), is one of the key steps in proteomic analysis. The current technology of protein digestion in proteomic analysis is time-consuming, tedious and not-automated due to the poor stability and autolysis of trypsin. To improve the protein digestion process, trypsin was immobilized and stabilized on polymer nanofibers entrapping superparamagnetic nanoparticles (magnetic nanofibers, NP-NFs). By electrospinning the homogeneous mixture of superparamagnetic nanoparticles (NPs) and polystyrene-poly(styrene-co-maleic anhydride), NPs could be effectively entrapped within polymer nanofibers, generating magnetically-separable nanofibers with high surface area for trypsin immobilization via the approach of enzyme coatings. Trypsin coatings on magnetic nanofibers (EC-TR/NP-NFs; EC-TR), fabricated via simple attachment of crosslinked trypsin molecules onto NP-NFs, were highly stable and could be recycled via facile magnetic separation. EC-TR showed negligible loss of trypsin activity even after incubation in an aqueous buffer under rigorous shaking (200. rpm) for 80. days, while the control samples of covalently-attached trypsin on NP-NFs (CA-TR/NP-NFs; CA-TR) and free trypsin lost more than 90% of their initial activities within 11 and 6. days, respectively. When highly-stable and magnetically-separable EC-TR was employed for the repetitive digestions of enolase under recycled uses for the duration of 50. days and even after treatment with another protease (chymotrypsin) for 32. h, the performance of enolase digestion was successfully maintained. The use of EC-TR for the enolase digestion in the ultra-sonication system resulted in fast (~10. min) and efficient digestions with reproducible performance under recycled uses.

Original language	English
Pages (from-to)	770-777
Number of pages	8
Journal	Chemical Engineering Journal
Volume	288
DOIs	https://doi.org/10.1016/j.cej.2015.12.003
Publication status	Published - 2016 Mar 15

Bibliographical note

Funding Information:
This work was supported by “International Collaborative R&D Program” and “Energy Efficiency & Resources Core Technology Program” of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grants funded by the Korea government Ministry of Trade, Industry and Energy ( 20118510020020 and 20142020200980 ). This work was also supported by the Global Research Laboratory Program ( 2014K1A1A2043032 ), Nano-Material Technology Development Program (2014M3A7B4052193), Converging Research Center Program (2013K000229) and Multi-omics Research Program ( NRF-2012M3A9B9036675 ) through the National Research Foundation of Korea ( NRF ) grant funded by the Korea government Ministry of Science, ICT and Future Planning ( MSIP ). B.C. Kim also acknowledges the Korea Institute of Science and Technology (KIST) research program ( 2E25290 ).

Publisher Copyright:
© 2015 Elsevier B.V.

Keywords

Enzyme immobilization and stabilization
Magnetic nanofibers
Nanobiocatalysis
Proteomics
Trypsin digestion
Ultra-sonication system

ASJC Scopus subject areas

General Chemistry
Environmental Chemistry
General Chemical Engineering
Industrial and Manufacturing Engineering

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10.1016/j.cej.2015.12.003

Cite this

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title = "Efficient protein digestion using highly-stable and reproducible trypsin coatings on magnetic nanofibers",

abstract = "Protein digestion, using an enzyme called trypsin (TR), is one of the key steps in proteomic analysis. The current technology of protein digestion in proteomic analysis is time-consuming, tedious and not-automated due to the poor stability and autolysis of trypsin. To improve the protein digestion process, trypsin was immobilized and stabilized on polymer nanofibers entrapping superparamagnetic nanoparticles (magnetic nanofibers, NP-NFs). By electrospinning the homogeneous mixture of superparamagnetic nanoparticles (NPs) and polystyrene-poly(styrene-co-maleic anhydride), NPs could be effectively entrapped within polymer nanofibers, generating magnetically-separable nanofibers with high surface area for trypsin immobilization via the approach of enzyme coatings. Trypsin coatings on magnetic nanofibers (EC-TR/NP-NFs; EC-TR), fabricated via simple attachment of crosslinked trypsin molecules onto NP-NFs, were highly stable and could be recycled via facile magnetic separation. EC-TR showed negligible loss of trypsin activity even after incubation in an aqueous buffer under rigorous shaking (200. rpm) for 80. days, while the control samples of covalently-attached trypsin on NP-NFs (CA-TR/NP-NFs; CA-TR) and free trypsin lost more than 90% of their initial activities within 11 and 6. days, respectively. When highly-stable and magnetically-separable EC-TR was employed for the repetitive digestions of enolase under recycled uses for the duration of 50. days and even after treatment with another protease (chymotrypsin) for 32. h, the performance of enolase digestion was successfully maintained. The use of EC-TR for the enolase digestion in the ultra-sonication system resulted in fast (~10. min) and efficient digestions with reproducible performance under recycled uses.",

keywords = "Enzyme immobilization and stabilization, Magnetic nanofibers, Nanobiocatalysis, Proteomics, Trypsin digestion, Ultra-sonication system",

author = "Byoungsoo Lee and Kim, {Byoung Chan} and Chang, {Mun Seock} and Kim, {Han Sol} and Na, {Hyon Bin} and Park, {Yong Il} and Jinwoo Lee and Taeghwan Hyeon and Hookeun Lee and Lee, {Sang Won} and Jungbae Kim",

note = "Funding Information: This work was supported by “International Collaborative R&D Program” and “Energy Efficiency & Resources Core Technology Program” of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grants funded by the Korea government Ministry of Trade, Industry and Energy ( 20118510020020 and 20142020200980 ). This work was also supported by the Global Research Laboratory Program ( 2014K1A1A2043032 ), Nano-Material Technology Development Program (2014M3A7B4052193), Converging Research Center Program (2013K000229) and Multi-omics Research Program ( NRF-2012M3A9B9036675 ) through the National Research Foundation of Korea ( NRF ) grant funded by the Korea government Ministry of Science, ICT and Future Planning ( MSIP ). B.C. Kim also acknowledges the Korea Institute of Science and Technology (KIST) research program ( 2E25290 ). Publisher Copyright: {\textcopyright} 2015 Elsevier B.V.",

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doi = "10.1016/j.cej.2015.12.003",

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journal = "Chemical Engineering Journal",

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T1 - Efficient protein digestion using highly-stable and reproducible trypsin coatings on magnetic nanofibers

AU - Lee, Byoungsoo

AU - Kim, Byoung Chan

AU - Chang, Mun Seock

AU - Kim, Han Sol

AU - Na, Hyon Bin

AU - Park, Yong Il

AU - Lee, Jinwoo

AU - Hyeon, Taeghwan

AU - Lee, Hookeun

AU - Lee, Sang Won

AU - Kim, Jungbae

N1 - Funding Information: This work was supported by “International Collaborative R&D Program” and “Energy Efficiency & Resources Core Technology Program” of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grants funded by the Korea government Ministry of Trade, Industry and Energy ( 20118510020020 and 20142020200980 ). This work was also supported by the Global Research Laboratory Program ( 2014K1A1A2043032 ), Nano-Material Technology Development Program (2014M3A7B4052193), Converging Research Center Program (2013K000229) and Multi-omics Research Program ( NRF-2012M3A9B9036675 ) through the National Research Foundation of Korea ( NRF ) grant funded by the Korea government Ministry of Science, ICT and Future Planning ( MSIP ). B.C. Kim also acknowledges the Korea Institute of Science and Technology (KIST) research program ( 2E25290 ). Publisher Copyright: © 2015 Elsevier B.V.

PY - 2016/3/15

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N2 - Protein digestion, using an enzyme called trypsin (TR), is one of the key steps in proteomic analysis. The current technology of protein digestion in proteomic analysis is time-consuming, tedious and not-automated due to the poor stability and autolysis of trypsin. To improve the protein digestion process, trypsin was immobilized and stabilized on polymer nanofibers entrapping superparamagnetic nanoparticles (magnetic nanofibers, NP-NFs). By electrospinning the homogeneous mixture of superparamagnetic nanoparticles (NPs) and polystyrene-poly(styrene-co-maleic anhydride), NPs could be effectively entrapped within polymer nanofibers, generating magnetically-separable nanofibers with high surface area for trypsin immobilization via the approach of enzyme coatings. Trypsin coatings on magnetic nanofibers (EC-TR/NP-NFs; EC-TR), fabricated via simple attachment of crosslinked trypsin molecules onto NP-NFs, were highly stable and could be recycled via facile magnetic separation. EC-TR showed negligible loss of trypsin activity even after incubation in an aqueous buffer under rigorous shaking (200. rpm) for 80. days, while the control samples of covalently-attached trypsin on NP-NFs (CA-TR/NP-NFs; CA-TR) and free trypsin lost more than 90% of their initial activities within 11 and 6. days, respectively. When highly-stable and magnetically-separable EC-TR was employed for the repetitive digestions of enolase under recycled uses for the duration of 50. days and even after treatment with another protease (chymotrypsin) for 32. h, the performance of enolase digestion was successfully maintained. The use of EC-TR for the enolase digestion in the ultra-sonication system resulted in fast (~10. min) and efficient digestions with reproducible performance under recycled uses.

AB - Protein digestion, using an enzyme called trypsin (TR), is one of the key steps in proteomic analysis. The current technology of protein digestion in proteomic analysis is time-consuming, tedious and not-automated due to the poor stability and autolysis of trypsin. To improve the protein digestion process, trypsin was immobilized and stabilized on polymer nanofibers entrapping superparamagnetic nanoparticles (magnetic nanofibers, NP-NFs). By electrospinning the homogeneous mixture of superparamagnetic nanoparticles (NPs) and polystyrene-poly(styrene-co-maleic anhydride), NPs could be effectively entrapped within polymer nanofibers, generating magnetically-separable nanofibers with high surface area for trypsin immobilization via the approach of enzyme coatings. Trypsin coatings on magnetic nanofibers (EC-TR/NP-NFs; EC-TR), fabricated via simple attachment of crosslinked trypsin molecules onto NP-NFs, were highly stable and could be recycled via facile magnetic separation. EC-TR showed negligible loss of trypsin activity even after incubation in an aqueous buffer under rigorous shaking (200. rpm) for 80. days, while the control samples of covalently-attached trypsin on NP-NFs (CA-TR/NP-NFs; CA-TR) and free trypsin lost more than 90% of their initial activities within 11 and 6. days, respectively. When highly-stable and magnetically-separable EC-TR was employed for the repetitive digestions of enolase under recycled uses for the duration of 50. days and even after treatment with another protease (chymotrypsin) for 32. h, the performance of enolase digestion was successfully maintained. The use of EC-TR for the enolase digestion in the ultra-sonication system resulted in fast (~10. min) and efficient digestions with reproducible performance under recycled uses.

KW - Enzyme immobilization and stabilization

KW - Magnetic nanofibers

KW - Nanobiocatalysis

KW - Proteomics

KW - Trypsin digestion

KW - Ultra-sonication system

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ER -

Efficient protein digestion using highly-stable and reproducible trypsin coatings on magnetic nanofibers

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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