1995 Fiscal Year Final Research Report Summary
Capillary HPLC/electrospray ionization mass spectrometry for structural analysis of micro-quantities of proteins
| Project/Area Number |
06680584
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Structural biochemistry
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| Research Institution | Osaka University |
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Principal Investigator |
TAKAO Toshifumi Inst.for Protein Res., Osaka Unv., Instructor, たんぱく質研究所, 助手 (10197048)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Capillary HPLC / Electrospray ionization / Magnetic sector mass spectrometer / Sensitive and accurate mass measurement / Reading frame / Modifications / Micro-blotting / Polyvinylidence fluoride membrane |
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| Research Abstract |
Capillary high-performance liquid chromatography (HPLC) has been combined with a magnetic sector mass spectrometer by improving the electrospray ion source. It allows the sensitive and accurate mass measurement in the HPLC/ESI-MS.As a result, all the components involved in a mixture sample such as an enzymatic digest of a protein could be efficiently measured following separation by a capillary column (300 mm diameter). Analyzes of a protein mixture and proteolytic digest indicate that the combination of a capillary HPLC and magnetic sector mass spectrometer is capable of attaining better than 0.01 % accuracy in mass determination on picomole quantities. It could successfully be applied to determination of the reading frames of DNA-derived protein sequence and both sites and types of modifications present in recombinant or natural proteins. A micro-blotting apparatus, which had been designed to be coupled with a capillary HPLC for collecting small volumes of sample solution eluted from a capillary column onto a polyvinylidene fluoride membrane, was demonstrated to fractionate each component separated by a capillary column with the high recovery (more than 90 %), which could readily be subjected to amino acid composition or sequence analyzes. The combination of a capillary HPLC/ESI-MS and micro-blotting system would go a good way for micro-analysis of peptides and proteins that are available only in limited quantities.
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Research Products
(14 results)
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[Publications] Takao, T., Tambara, Y., Nakamura, A., Yoshino, K., Fukuda, M.& Shimonishi, Y.: "Sensitive Analysis of Oligosaccharides Derivatized with 4-Aminobenzoic Acid 2- (Diethylamino) ethyl Ester by Matri x-Assisted Laser Desorption/Ionization Mass Spectrometry" Rapid Commun.Mass Spectrom. (accepted). (1996)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Shimizu, T., Hozumi, K., Horiike, S., Nunomura, K., Ikegami, S., Takao, T., & Shimonishi, Y.: "A Covalently Cross-Linked Histone Heterodimer in Starfish" Nature. 380. 32 (1996)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Gonzalez, J., Besada, V., Garay, H., Reyes, O., Padron, G., Tambara, Y., Takao, T., & Shimonishi, Y.: "Effect of the Position of a Basic Amino Acid on C-Terminal Rearrangement of Protonated Peptides Upon Collision-induced Dissociation." J.Mass Spectrom.31. 150-158 (1996)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Yamada, T., Uyeda, A., Takao, T., Shimonishi, Y., Matsushima, M., & Kikuchi, M.: "O-Glycosylation of the Thr^<70> Residue of Cell Adhesive Lysozyme in Yeast" Eur.J.Biochem.230. 965-970 (1995)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Yoshino, K., Takao, T., Huang, X., Murata, H., Nakao, H., Takeda, T., & Shimonishi, Y.: "Characterization of a Highly Toxic, Large Molecular Size Heat-Stable Enterotoxin Produced by a Clinucal Isolate of Yersinia enterocolitica." FEBS Lett.362. 319-322 (1995)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Daquinag, A.C., Nakamura, S., Takao, T., Shimonishi, Y., & Tsukamoto, T.: "Primary Structure of a Novel Potent Endogenous DOPA-Containing Inhibitor of Phenoloxidase from Musca domestica." Proc.Natl.Acad.Sci., U.S.A.92. 2964-2968 (1995)
Description
「研究成果報告書概要(欧文)」より
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