Limited Proteolysis in Proteomics Using Protease-Immobilized Microreactors

  • Hiroshi Yamaguchi
  • Masaya MiyazakiEmail author
  • Hideaki Maeda
Part of the Methods in Molecular Biology book series (MIMB, volume 815)


Proteolysis is the key step for proteomic studies integrated with MS analysis. Compared with the conventional method of in-solution digestion, proteolysis by a protease-immobilized microreactor has a number of advantages for proteomic analysis; i.e., rapid and efficient digestion, elimination of a purification step of the digests prior to MS, and high stability against a chemical or thermal denaturant. This chapter describes the preparation of the protease-immobilized microreactors and proteolysis performance of these microreactors. Immobilization of proteases by the formation of a polymeric membrane consisting solely of protease-proteins on the inner wall of the microchannel is performed. This was realized either by a cross-linking reaction in a laminar flow between lysine residues sufficiently present on the protein surfaces themselves or in the case of acidic proteins by mixing them with poly-lysine prior to the crosslink-reaction. The present procedure is simple and widely useful not only for proteases but also for several other enzymes.

Key words

Enzyme immobilization Microfluidics Microreactor Protease Proteolysis Proteomics 



The authors thank Dr. T. Honda for carrying out the initial experiments. Part of this work was supported by Grant-in-Aid for Basic Scientific Research (B: 20310074 and 23310092) from JSPS.


  1. 1.
    Aebersold, R, Mann, M. (2003) Mass spectrometry-based proteomics, Nature 422, 198–207Google Scholar
  2. 2.
    Domon, B., Aebersold, R. (2006) Mass spectrometry and protein analysis, Science 312, 212–217Google Scholar
  3. 3.
    Witze, E. S., Old, W. N., Resing, K. A., Ahn, N. G. (2007) Mapping protein post-translational modifications with mass spectrometry, Nat. Methods 10, 798–806Google Scholar
  4. 4.
    Liu, Y., Liu, B., Yang, P., Girault, H. H., (2008) Microfluidic enzymatic reactors for proteome research, Anal. Bioanal. Chem. 390, 227–229Google Scholar
  5. 5.
    Ma, J., Zhang, L., Liang, Z., Zhang, W., Zhang, Y. (2009) Recent advance in immobilized enzymatic reactors and their applications in proteome analysis, Anal. Chim. Acta 632, 1–8Google Scholar
  6. 6.
    Miyazaki, M., Maeda, H. (2006) Microchannel enzyme reactors and their applications for processing, Trends Biotechnol. 24, 463–470Google Scholar
  7. 7.
    Miyazaki, M., Honda, T., Yamaguchi, H., Briones, M. P. P., Maeda, H. (2008) in: Harding S. E. (Eds.), Biotechnology & Genetic Engineering Reviews, Nottingham University Press, Nottingham, 25, 405–428Google Scholar
  8. 8.
    Ma, J., Zhang, L., Liang, Z., Zhang, W., Zhang, Y. (2007) Monolith-based immobilized enzyme reactors: Recent developments and applications for proteome analysis, J. Sep. Sci. 30, 3050–3059Google Scholar
  9. 9.
    Honda, T., Miyazaki, M., Nakamura, H., Maeda, H. (2005) Immobilization of enzymes on a microchannel surface through cross-linking polymerization, Chem. Commun. 5062–5064Google Scholar
  10. 10.
    Honda, T., Miyazaki, M., Nakamura, H., Maeda, H. (2006) Facile preparation of an enzyme-immobilized microreactor using a cross-linking enzyme membrane on a microchannel surface, Adv. Synth. Catal. 348, 2163–2171Google Scholar
  11. 11.
    Ma, J., Ziang, 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, Anal. Chem. 80, 2949–2956Google Scholar
  12. 12.
    Yamaguchi, H., Miyazaki, M., Honda, T., Briones-Nagata, M. P., Arima, K., Maeda, H. (2009) Rapid and efficient proteolysis for proteomic analysis by protease-immobilized microreactor, Electrophoresis 30, 3257–3264Google Scholar
  13. 13.
    Yamaguchi, H., Miyazaki, M., Kawazumi, H., Maeda, H. (2010) Multidigestion in continuous flow tandem protease-immobilized microreactors for proteomic analysis. Anal. Biochem. 407, 12–18Google Scholar
  14. 14.
    Park, Z. -Y., Russell, D. H. (2000) Thermal denaturation: A useful technique in peptide mass mapping. Anal. Chem. 72, 2667–2670Google Scholar
  15. 15.
    Sim, T. S., Kim, E. -M., Joo, H. S., Kim, B. G., Kim, Y. -K. (2006) Application of a temperature-controllable microreactor to simple and rapid protein identification using MALDI-TOF MS. Lab. Chip 6, 1056–1061Google Scholar
  16. 16.
    Liu, T., Bao, H., Zhang, L., Chen, G. (2009) Integration of electrodes in a suction cup-driven microchip for alternating current-accelerated proteolysis. Electrophoresis 30, 3265–3268Google Scholar
  17. 17.
    Yamaguchi, H., Miyazaki, M., Maeda, H. (2010) Proteolysis approach without chemical modification for a simple and rapid analysis of disulfide bonds using thermostable protease-immobilized microreactors. Proteomics 10, 2942–2949Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Hiroshi Yamaguchi
    • 1
  • Masaya Miyazaki
    • 2
    Email author
  • Hideaki Maeda
    • 1
  1. 1.Measurement Solution Research CenterNational Institute of Advanced Industrial Science and TechnologyTosuJapan
  2. 2.Measurement Solution Research CenterNational Institute of Advanced Industrial Science and TechnologyTosuJapan

Personalised recommendations