title: Proteolysis approach without chemical modification for a simple and rapid analysis of disulfide bonds using thermostable protease-immobilized microreactors.
abstract: Disulfide bonds in proteins are important not only for the conformational stability of the protein but also for the regulation of oxidation-reduction in signal transduction. The conventional method for the assignment of disulfide bond by chemical cleavage and/or proteolysis is a time-consuming multi-step procedure. In this study, we report a simple and rapid analysis of disulfide bond from protein digests that were prepared by the thermostable protease-immobilized microreactors. The feasibility and performance of this approach were evaluated by digesting lysozyme and BSA at several temperatures. The proteins which stabilize their conformations by disulfide bonds were thermally denatured during proteolysis and were efficiently digested by the immobilized protease but not by free protease. The digests were directly analyzed by ESI-TOF MS without any purification or concentration step. All four disulfide bonds on lysozyme and 10 of 17 on BSA were assigned from the digests by the trypsin-immobilized microreactor at 50 degrees C. The procedure for proteolysis and the assignment were achieved within 2 h without any reduction and alkylation procedure. From the present results, the proteolysis approach by the thermostable protease-immobilized microreactor provides a strategy for the high-throughput analysis of disulfide bond in proteomics.
authors.affiliation: National Institute of Advanced Industrial Science and Technology, Tosu, Saga, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Masaya
authors.initials: M
authors.lastname: Miyazaki
authors.forename: Hideaki
authors.initials: H
authors.lastname: Maeda
mesh_terms: D000818:Animals; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D002417:Cattle; D004220:Disulfides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
chemical_list: D004220:Disulfides; D004800:Enzymes, Immobilized; D012710:Serum Albumin, Bovine; D009113:Muramidase; D010447:Peptide Hydrolases