Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Apr;81(7):1956–1960. doi: 10.1073/pnas.81.7.1956

Strategy for the mass spectrometric verification and correction of the primary structures of proteins deduced from their DNA sequences.

B W Gibson, K Biemann
PMCID: PMC345415  PMID: 6371805

Abstract

Fast atom bombardment mass spectrometry has been used to confirm and correct regions from the amino acid sequences of three large proteins, glutaminyl- and glycyl-tRNA synthetase from Escherichia coli and methionyl-tRNA synthetase from yeast, whose primary structures had been deduced from the base sequences of their corresponding genes. The strategy is based on a comparison of the molecular weights of the tryptic peptides predicted from all three reading frames of the gene sequences with those determined mass spectrometrically. The experimental molecular weights either match or differ and can be used to assess the correctness of the base sequences, identify errors that lead to frame shifts, premature stop codons, incorrect amino acids, etc., or identify the presence of posttranslational modifications. This method is very fast and requires little material (5-20 nmol).

Full text

PDF
1959

Images in this article

1957Image
on p.1957

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Carnegie P. R. Digestion of an arg-pro bond by trypsin in the ecephalitogenic basic protein of human myelin. Nature. 1969 Aug 30;223(5209):958–959. doi: 10.1038/223958a0. [DOI] [PubMed] [Google Scholar]
  2. Chou J., Casadaban M. J., Lemaux P. G., Cohen S. N. Identification and characterization of a self-regulated repressor of translocation of the Tn3 element. Proc Natl Acad Sci U S A. 1979 Aug;76(8):4020–4024. doi: 10.1073/pnas.76.8.4020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Czernilofsky A. P., Levinson A. D., Varmus H. E., Bishop J. M., Tischer E., Goodman H. M. Nucleotide sequence of an avian sarcoma virus oncogene (src) and proposed amino acid sequence for gene product. Nature. 1980 Sep 18;287(5779):198–203. doi: 10.1038/287198a0. [DOI] [PubMed] [Google Scholar]
  4. Czernilofsky A. P., Levinson A. D., Varmus H. E., Bishop J. M., Tischer E., Goodman H. Corrections to the nucleotide sequence of the src gene of Rous sarcoma virus. Nature. 1983 Feb 24;301(5902):736–738. doi: 10.1038/301736b0. [DOI] [PubMed] [Google Scholar]
  5. Fullmer C. S., Wasserman R. H. Analytical peptide mapping by high performance liquid chromatography. Application to intestinal calcium-binding proteins. J Biol Chem. 1979 Aug 10;254(15):7208–7212. [PubMed] [Google Scholar]
  6. Heffron F., McCarthy B. J., Ohtsubo H., Ohtsubo E. DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3. Cell. 1979 Dec;18(4):1153–1163. doi: 10.1016/0092-8674(79)90228-9. [DOI] [PubMed] [Google Scholar]
  7. Hoben P., Royal N., Cheung A., Yamao F., Biemann K., Söll D. Escherichia coli glutaminyl-tRNA synthetase. II. Characterization of the glnS gene product. J Biol Chem. 1982 Oct 10;257(19):11644–11650. [PubMed] [Google Scholar]
  8. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Putney S. D., Royal N. J., Neuman de Vegvar H., Herlihy W. C., Biemann K., Schimmel P. Primary structure of a large aminoacyl-tRNA synthetase. Science. 1981 Sep 25;213(4515):1497–1501. doi: 10.1126/science.7025207. [DOI] [PubMed] [Google Scholar]
  11. Sanger F., Coulson A. R. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol. 1975 May 25;94(3):441–448. doi: 10.1016/0022-2836(75)90213-2. [DOI] [PubMed] [Google Scholar]
  12. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  13. Tarr G. E. Improved manual sequencing methods. Methods Enzymol. 1977;47:335–357. doi: 10.1016/0076-6879(77)47036-8. [DOI] [PubMed] [Google Scholar]
  14. Walter P., Gangloff J., Bonnet J., Boulanger Y., Ebel J. P., Fasiolo F. Primary structure of the Saccharomyces cerevisiae gene for methionyl-tRNA synthetase. Proc Natl Acad Sci U S A. 1983 May;80(9):2437–2441. doi: 10.1073/pnas.80.9.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Webster T. A., Gibson B. W., Keng T., Biemann K., Schimmel P. Primary structures of both subunits of Escherichia coli glycyl-tRNA synthetase. J Biol Chem. 1983 Sep 10;258(17):10637–10641. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES