Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Mar;177(5):1402–1404. doi: 10.1128/jb.177.5.1402-1404.1995

Accurate determination of the molecular weight of the major surface layer protein isolated from Clostridium thermosaccharolyticum by time-of-flight mass spectrometry.

G Allmaier 1, C Schäffer 1, P Messner 1, U Rapp 1, F J Mayer-Posner 1
PMCID: PMC176751  PMID: 7868619

Abstract

Matrix-assisted laser desorption with concomitant ionization, in combination with a linear time-of-flight mass spectrometer, was used to analyze underivatized and hard-to-solubilize surface layer proteins and glycoproteins by depositing them on top of a microcrystalline layer of the matrix alpha-cyano-4-hydroxycinnamic acid. Use of this special sample preparation technique allowed the first successful desorption-ionization of intact surface layer proteins and accurate determination of their molecular weights by mass spectrometry. The molecular mass of the monomeric subunit of the major surface layer protein isolated from Clostridium thermosaccharolyticum E207-71 was determined to be 75,621 +/- 81 Da. The obtainable mass accuracy of the technique is conservatively considered to be within +/- 0.2%. This result deviates from that given by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by approximately 7.4 kDa because this method is strongly affected and biased by the three-dimensional structure of this type of surface protein. With the apparent advantages of unsurpassed mass accuracy, low dependence on the physicochemical properties of the surface layer proteins, and high sensitivity, it can be concluded that a linear time-of-flight instrument combined with UV matrix-assisted laser desorption with concomitant ionization is better suited for molecular weight determination than is gel electrophoresis.

Full Text

The Full Text of this article is available as a PDF (174.5 KB).

Selected References

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

  1. Baumeister W., Wildhaber I., Engelhardt H. Bacterial surface proteins. Some structural, functional and evolutionary aspects. Biophys Chem. 1988 Feb;29(1-2):39–49. doi: 10.1016/0301-4622(88)87023-6. [DOI] [PubMed] [Google Scholar]
  2. Beavis R. C., Chait B. T. Cinnamic acid derivatives as matrices for ultraviolet laser desorption mass spectrometry of proteins. Rapid Commun Mass Spectrom. 1989 Dec;3(12):432–435. doi: 10.1002/rcm.1290031207. [DOI] [PubMed] [Google Scholar]
  3. Beavis R. C., Chait B. T. Rapid, sensitive analysis of protein mixtures by mass spectrometry. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6873–6877. doi: 10.1073/pnas.87.17.6873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hillenkamp F., Karas M., Beavis R. C., Chait B. T. Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers. Anal Chem. 1991 Dec 15;63(24):1193A–1203A. doi: 10.1021/ac00024a002. [DOI] [PubMed] [Google Scholar]
  5. Hollaus F., Sleytr U. On the taxonomy and fine structure of some hyperthermophilic saccharolytic Clostridia. Arch Mikrobiol. 1972;86(2):129–146. doi: 10.1007/BF00413368. [DOI] [PubMed] [Google Scholar]
  6. Karas M., Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct 15;60(20):2299–2301. doi: 10.1021/ac00171a028. [DOI] [PubMed] [Google Scholar]
  7. Lechner J., Sumper M. The primary structure of a procaryotic glycoprotein. Cloning and sequencing of the cell surface glycoprotein gene of halobacteria. J Biol Chem. 1987 Jul 15;262(20):9724–9729. [PubMed] [Google Scholar]
  8. Messner P., Sleytr U. B. Asparaginyl-rhamnose: a novel type of protein-carbohydrate linkage in a eubacterial surface-layer glycoprotein. FEBS Lett. 1988 Feb 15;228(2):317–320. doi: 10.1016/0014-5793(88)80023-1. [DOI] [PubMed] [Google Scholar]
  9. Messner P., Sleytr U. B. Bacterial surface layer glycoproteins. Glycobiology. 1991 Dec;1(6):545–551. doi: 10.1093/glycob/1.6.545. [DOI] [PubMed] [Google Scholar]
  10. Messner P., Sleytr U. B. Crystalline bacterial cell-surface layers. Adv Microb Physiol. 1992;33:213–275. doi: 10.1016/s0065-2911(08)60218-0. [DOI] [PubMed] [Google Scholar]
  11. Mock K. K., Sutton C. W., Cottrell J. S. Sample immobilization protocols for matrix-assisted laser-desorption mass spectrometry. Rapid Commun Mass Spectrom. 1992 Apr;6(4):233–238. doi: 10.1002/rcm.1290060402. [DOI] [PubMed] [Google Scholar]
  12. Sleytr U. B., Messner P. Crystalline surface layers in procaryotes. J Bacteriol. 1988 Jul;170(7):2891–2897. doi: 10.1128/jb.170.7.2891-2897.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sleytr U. B., Messner P. Crystalline surface layers on bacteria. Annu Rev Microbiol. 1983;37:311–339. doi: 10.1146/annurev.mi.37.100183.001523. [DOI] [PubMed] [Google Scholar]
  14. Sleytr U. B., Messner P., Pum D., Sára M. Crystalline bacterial cell surface layers. Mol Microbiol. 1993 Dec;10(5):911–916. doi: 10.1111/j.1365-2958.1993.tb00962.x. [DOI] [PubMed] [Google Scholar]
  15. Sumper M., Berg E., Mengele R., Strobel I. Primary structure and glycosylation of the S-layer protein of Haloferax volcanii. J Bacteriol. 1990 Dec;172(12):7111–7118. doi: 10.1128/jb.172.12.7111-7118.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES