Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion

Byoung Chan Kim, Daniel Lopez-Ferrer, Sang Mok Lee, Hye Kyung Ahn, Sujith Nair, Seong Kim, Beom Soo Kim, Konstantinos Petritis, David G. Camp, Jay W. Grate, Richard D. Smith, Yoon Mo Koo, Man Bock Gu, Jungbae Kim

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

A stable and robust trypsin-based biocatalytic system was developed and demonstrated for pro-teomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in "real-world" proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

Original languageEnglish (US)
Pages (from-to)1893-1900
Number of pages8
JournalProteomics
Volume9
Issue number7
DOIs
StatePublished - Apr 1 2009

Fingerprint

Nanofibers
Trypsin
Proteolysis
Polymers
Coatings
Proteins
Digestion
Proteomics
Autolysis
Chymotrypsin
Recycling
Glutaral
Proteome
Immobilization
Yeast

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Kim, B. C., Lopez-Ferrer, D., Lee, S. M., Ahn, H. K., Nair, S., Kim, S., ... Kim, J. (2009). Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion. Proteomics, 9(7), 1893-1900. https://doi.org/10.1002/pmic.200800591
Kim, Byoung Chan ; Lopez-Ferrer, Daniel ; Lee, Sang Mok ; Ahn, Hye Kyung ; Nair, Sujith ; Kim, Seong ; Kim, Beom Soo ; Petritis, Konstantinos ; Camp, David G. ; Grate, Jay W. ; Smith, Richard D. ; Koo, Yoon Mo ; Gu, Man Bock ; Kim, Jungbae. / Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion. In: Proteomics. 2009 ; Vol. 9, No. 7. pp. 1893-1900.
@article{a84174708a174f4ca851897156913f1b,
title = "Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion",
abstract = "A stable and robust trypsin-based biocatalytic system was developed and demonstrated for pro-teomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in {"}real-world{"} proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.",
author = "Kim, {Byoung Chan} and Daniel Lopez-Ferrer and Lee, {Sang Mok} and Ahn, {Hye Kyung} and Sujith Nair and Seong Kim and Kim, {Beom Soo} and Konstantinos Petritis and Camp, {David G.} and Grate, {Jay W.} and Smith, {Richard D.} and Koo, {Yoon Mo} and Gu, {Man Bock} and Jungbae Kim",
year = "2009",
month = "4",
day = "1",
doi = "10.1002/pmic.200800591",
language = "English (US)",
volume = "9",
pages = "1893--1900",
journal = "Proteomics",
issn = "1615-9853",
publisher = "Wiley-VCH Verlag",
number = "7",

}

Kim, BC, Lopez-Ferrer, D, Lee, SM, Ahn, HK, Nair, S, Kim, S, Kim, BS, Petritis, K, Camp, DG, Grate, JW, Smith, RD, Koo, YM, Gu, MB & Kim, J 2009, 'Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion', Proteomics, vol. 9, no. 7, pp. 1893-1900. https://doi.org/10.1002/pmic.200800591

Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion. / Kim, Byoung Chan; Lopez-Ferrer, Daniel; Lee, Sang Mok; Ahn, Hye Kyung; Nair, Sujith; Kim, Seong; Kim, Beom Soo; Petritis, Konstantinos; Camp, David G.; Grate, Jay W.; Smith, Richard D.; Koo, Yoon Mo; Gu, Man Bock; Kim, Jungbae.

In: Proteomics, Vol. 9, No. 7, 01.04.2009, p. 1893-1900.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion

AU - Kim, Byoung Chan

AU - Lopez-Ferrer, Daniel

AU - Lee, Sang Mok

AU - Ahn, Hye Kyung

AU - Nair, Sujith

AU - Kim, Seong

AU - Kim, Beom Soo

AU - Petritis, Konstantinos

AU - Camp, David G.

AU - Grate, Jay W.

AU - Smith, Richard D.

AU - Koo, Yoon Mo

AU - Gu, Man Bock

AU - Kim, Jungbae

PY - 2009/4/1

Y1 - 2009/4/1

N2 - A stable and robust trypsin-based biocatalytic system was developed and demonstrated for pro-teomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in "real-world" proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

AB - A stable and robust trypsin-based biocatalytic system was developed and demonstrated for pro-teomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in "real-world" proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

UR - http://www.scopus.com/inward/record.url?scp=65349150524&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=65349150524&partnerID=8YFLogxK

U2 - 10.1002/pmic.200800591

DO - 10.1002/pmic.200800591

M3 - Article

VL - 9

SP - 1893

EP - 1900

JO - Proteomics

JF - Proteomics

SN - 1615-9853

IS - 7

ER -