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Deblocking and Subsequent Microsequence Analysis of N-Terminally Blocked Proteins Immobilized on PVDF Membrane

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Methods in Protein Sequence Analysis

Abstract

In recent protein microsequence analyses, proteins separated by polyacrylamide gel electrophoresis (PAGE) and electroblotted onto a polyvinylidene difluoride (PVDF) membrane can be easily sequenced by a gas-phase sequencer (Matsudaira, 1987). However, even if the proteins are successfully separated by PAGE and electroblotted onto PVDF membrane, the N-terminal amino acid sequences of proteins with a blocking group at the N-terminus cannot be determined by Edman degradation. A large number of proteins are known to be N-terminally blocked (Aitken, 1990; Tsunasawa and Sakiyama, 1992). For example, 51 of 72 seed proteins in the rice and 14 of 36 seed proteins in the winged bean which were separated by two-dimensional gel electrophoresis were estimated to be N-terminally blocked (Hirano, 1989; Komatsu et al., 1992). It is, therefore, important to establish simple and rapid deblocking and sequencing techniques for these proteins.

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References

  • Aitken, A., 1990 Identification of Proteins Consensus Sequences, Ellis Horwood Ltd., Hemel Hempstead, UK.

    Google Scholar 

  • Fames, T. C., Harris, A., Auffret, A. D. and Aitken, A., 1991 Removal of N-acetyl groups from blocked peptides with acylpeptide hydrolase, Eur. J. Biochem. 196:679.

    Article  Google Scholar 

  • Hirano, H., 1989 Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel, J. Protein Chem. 8:115.

    Article  PubMed  CAS  Google Scholar 

  • Hirano, H., Komatsu, S., Kajiwara, H., Takakura, H, Sakiyama, F. and Tsunasawa, S., 1991a Microsequence analysis of N α-blocked proteins electroblotted onto an immobilizing matrix from polyacrylamide gels, Anal. Sci. 7:suppl. 941.

    Article  CAS  Google Scholar 

  • Hirano, H., Komatsu, S., Nakamura, A., Kikuchi, F., Kajiwara, H., Tsunasawa, S. and Sakiyama, F., 1991b Structural homology between semidwarfism-related proteins and glutelin seed protein in rice (Oryza sativa L.), Theor. Appl. Genet. 83:153.

    Article  CAS  Google Scholar 

  • Hirano, H., Komatsu, S., Takakura, H., Sakiyama, F. and Tsunasawa, S., 1992 Deblocking and subsequent microsequence analysis of N α-blocked proteins electroblotted onto PVDF membrane, J. Biochem. 111:754.

    PubMed  CAS  Google Scholar 

  • Hirano, H, and Watanabe, T., 1990 Micro sequencing of proteins electroblotted onto immobilizing matrices from polyacrylamide gel electrophoresis: Application to an insoluble protein. Electrophoresis 11:573.

    Article  PubMed  CAS  Google Scholar 

  • Ikeuchi, M. and Inoue, Y., 1988 A new photosystem II reaction center component (4.8 kDa protein) encoded by chloroplast genome, FEBS Lett. 241:99.

    Article  PubMed  CAS  Google Scholar 

  • Komatsu, S. and Hirano, H., 1992 Rice seed globulin: A protein similar to wheat seed glutelin, Phytochemistry 31:3455.

    Article  PubMed  CAS  Google Scholar 

  • Komatsu, S., Kajiwara, H. and Hirano, H., 1992 Rice protein library: a data file of rice proteins separated by two-dimensional electrophoresis, Nippon Nogeikagaku Kaishi 66:166.

    Google Scholar 

  • Krishna, R. G., Chin, C. C. Q. and Wold, F., 1991 N-terminal sequence analysis of N-acetylated proteins after unblocking with N-acylaminoacyl-peptide hydrolase, Anal. Biochem. 199:45.

    Article  PubMed  CAS  Google Scholar 

  • Matsudaira, P., 1987 Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes, J. Biol. Chem. 262:10035.

    PubMed  CAS  Google Scholar 

  • Moerschell, R. P., Hosokawa, Y., Tsunasawa, S. and Sherman, F., 1990 The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo. Processing of altered iso-1-cytochrome c created by oligonucleotide transformation. J. Biol. Chem. 265:19638.

    PubMed  CAS  Google Scholar 

  • Moyer, M., Harper, A., Payne, G., Ryals, J. and Fowler, E., 1990 In situ digestion with pyroglutamate aminopeptidase for N-terminal sequencing electroblotted proteins. J. Protein Chem. 9:282.

    Google Scholar 

  • Podell, D. N. and Abraham, G. N., 1978 A technique for the removal of pyroglutamic acid from the amino terminus of proteins using calf liver pyroglutamyl amino peptidase, Biochem. Biophys. Res. Commun. 81:176.

    Article  PubMed  CAS  Google Scholar 

  • Sheehan, J. C. and Yang, D. D. H., 1958 The use of N-formylamino acids in peptide synthesis, J. Am. Chem. Soc. 80:1154.

    Article  CAS  Google Scholar 

  • Sugino, Y., Tsunasawa, S., Yutani, K., Ogasahara, K. and Suzuki, M., 1980 Amino-terminally formylated tryptophan synthase α-subunit produced by the TRP operon cloned in a plasmid vector, J. Biochem. 87:351.

    PubMed  CAS  Google Scholar 

  • Tsunasawa, S. and Sakiyama, F., 1992 Amino-terminal acetylation of proteins, in: The Post-translational Modification of Proteins, Tuboi, S., Taniguchi, N. and Katsunuma, N., eds. 113.

    Google Scholar 

  • Tsunasawa, S. and Hirano, H., 1992 Microsequence analysis of N-terminally blocked proteins, J. Protein Chem. 11:382.

    Article  Google Scholar 

  • Tsunasawa, S., Takakura, H. and Sakiyama, F., 1990 Microsequence analysis of N-acetylated proteins, J. Protein Chem. 9:265.

    Google Scholar 

  • Wellner, D., Panneerselvam, C. and Horecker, B. L., 1990 Sequencing of peptides and proteins with blocked N-terminal amino acids: N-Acetylserine or N-acetylthreonine, Proc. Natl. Acad. Sci. USA 87:1947.

    Article  PubMed  CAS  Google Scholar 

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© 1993 Springer Science+Business Media New York

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Tsunasawa, S., Hirano, H. (1993). Deblocking and Subsequent Microsequence Analysis of N-Terminally Blocked Proteins Immobilized on PVDF Membrane. In: Imahori, K., Sakiyama, F. (eds) Methods in Protein Sequence Analysis. Springer, Boston, MA. https://doi.org/10.1007/978-1-4899-1603-7_6

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  • DOI: https://doi.org/10.1007/978-1-4899-1603-7_6

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-1-4899-1605-1

  • Online ISBN: 978-1-4899-1603-7

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