Capillary HPLC/electrospray ionization mass spectrometry for structural analysis of micro-quantities of proteins

• Project/Area Number: 06680584
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: Structural biochemistry
• Research Institution: Osaka University
• Principal Investigator: TAKAO Toshifumi Inst.for Protein Res., Osaka Unv., Instructor, たんぱく質研究所, 助手 (10197048)
• Project Period (FY): 1994 – 1995
• Project Status: Completed (Fiscal Year 1995)
• Budget Amount: ¥2,000,000 (Direct Cost: ¥2,000,000); Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000); Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
• Keywords: Capillary HPLC / Electrospray ionization / Magnetic sector mass spectrometer / Sensitive and accurate mass measurement / Reading frame / Modifications / Micro-blotting / Polyvinylidence fluoride membrane / 蛋白質の一次構造解析 / 安定同位体 / カルボキシ末端ペプチド / 微量試量 / イオン化効率 / 分子量測定 / 蛋白質のブロッティング

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.

Research Products

• [Publications] Takao, T.: "Sensitive Analysis of Oligosaccharides Derivatized with 4-Aminobenzoic Acid 2-(Diethylamino) ethyl Ester by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry" Rapid Commun. Mass Spectrom.(accepted). (1996)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Shimizu, T.: "A Covalently Cross-Linked Histone Heterodimer in Starfish" Nature. 380. (1996)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Conzalez, J.: "Effect of the Position of a Basic Amino Acid on C-Terminal Rearrangement of Protonated Peptides Upon Collision-induced Dissociation" J. Mass Spectrom.(in press). (1996)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Yoshino, K.: "Use of the Derivatizing Agent, 4-Aminobenzoic Acid 2-(Diethyl-amino) ethyl Ester for High-Sensitivity Detection of Oligo-saccharides by Electrospray Ionization Mass Spectrometry" Anal. Chem.67. 4028-4031 (1995)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Yamada, T.: "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(和文)」より
• [Publications] Yoshino, K.: "Characterization of Highly Toxic, Large Molecular Size Heat-Stable Enterotoxin Produced by a Clinical Isolate of Yersinia enterocolitica" FEBS Lett.362. 319-322 (1995)
  • Description: 「研究成果報告書概要(和文)」より
• [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
• [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
• [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
• [Publications] Yoshino, K., Takao, T., Murata, H., & Shimonishi, Y.: "Use of the Derivatizing Agent, 4-Aminobenzoic Acid 2- (Diethylamino) ethyl Ester for High-Sensitivity Detection of Oligosaccharides by Electrospray lonization Mass Spectrometry." Anal.Chem.67. 4028-4031 (1995)
  • Description: 「研究成果報告書概要(欧文)」より
• [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
• [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
• [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
• [Publications] Murata, H., Takao, T., & Shimonishi, Y.: "Capillary Liquid Chromatography/Electrospray Mass Spectrometry with a Magnetic Sector Instrument for Accurate Mass Measurement of Peptide and Protein Mixtures." "Mass Spectrometry in the Biological Sciences", Eds.Burlingame, A & Carr, S.A..Humana. 267-280 (1995)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Yoshino,K.: "Use of the Derivatizing Agent,4-Aminobenzoic Acid 2-(Diethylamino) ethyl Ester for High-Sensitivity Detection of Oligosaccharides by Etectrospray Ioniation Mass Spectrometry." Anal.Chem.67. 4028-4031 (1995)
• [Publications] Imamoto,N.: "In Vivo Evidence for Involvement of a 58 kDa Component of Nuclear Pore-Targeting Complex in Nuclear Protein Import." EMBO,J.14. 3617-3626 (1995)
• [Publications] Imamoto,N.: "The Nuclear Pore-Targeting Complex Binds to Nuclear Pores after Association with a Karyophile." FEBS Lett.368. 415-419 (1995)
• [Publications] Sanada,K.: "Role of Heterogeneous N-Terminal Acylation of Recoverin in Rhodopsin Phosphorylation." J.Biol.Chem.270. 15459-15462 (1995)
• [Publications] Daquinag,A.C.: "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)
• [Publications] Murata,H.: "Capillary Liquid Chromatography/Electrospray Mass Spectrometry with a Magnetic Sector Instrument for Accurate Mass Measurement of Peptide and Protein Mixtures." Elsevier (NewYork). 267-280 (1995)
• [Publications] Yoshino,K.: "Purification and Characterization of a Novel Superantigen Produced by a Clinical Isolate of Yersinia pseudotuberculosis." FEBS Lett.356. 141-144 (1994)
• [Publications] Takao,T.: "Positive-Ion Fast Atom Bombardment Tandem Mass Spectrometry of Peptide Nucleic Acids." Rapid Commun.Mass Spectrom.8. 925-928 (1994)
• [Publications] Matsuda,T.: "Characterization of Interactions between Transducin α/Br-Subunits and Lipid Membrans." J.Biol.Chem. 269. 30358-30363 (1994)
• [Publications] Agawara,K.: "Activation Peptide of Human Factor IX Has Oligosaccharides O-Glycosidically Linked to Threonine residues at 159 and 169" Biochemistry. 33. 5167-5171 (1994)
• [Publications] Murata,H.: "Optimization of Skimmer Voltages of an Electrospray Ion Source Coupled with a Magnetic Sector Instrument." Rapid Commun.Mass Spectrom.8. 205-210 (1994)
• [Publications] Fukada,Y.: "Effects of Carboxyl Methylation of Photoreceptor G Protein γ-Subunit in Visual Transducin." Biol.Chem.269. 5163-5170 (1994)