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Limited Proteolysis in Proteomics Using Protease-Immobilized Microreactors

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

Abstract

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 

Notes

Acknowledgments

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.

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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

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