title: Limited proteolysis in proteomics using protease-immobilized microreactors.
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.
authors: Measurement Solution Research Center, National Institute of Advanced Industrial Science and Technology, Tosu, Saga, Japan.
authors: Hiroshi
authors: H
authors: Yamaguchi
authors: Masaya
authors: M
authors: Miyazaki
authors: Hideaki
authors: H
authors: Maeda
mesh_terms: D002918:Chymotrypsin / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006868:Hydrolysis; D013058:Mass Spectrometry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry; D059748:Proteolysis; D040901:Proteomics; D014357:Trypsin / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011107:Polylysine; D002918:Chymotrypsin; D014357:Trypsin