Direct Mass Spectrometric Analyses for Protein Chemistry Studies

  • Scott D. Buckel
  • Tracy I. Stevenson
  • Joseph A. Loo

Abstract

For structural studies of various proteins, a combination of traditional sequence analysis and mass spectrometry (MS) has been effectively used in our laboratory. Protein sequencing methods and mass spectrometry are often plagued with the same problems in that samples are frequently contaminated with other materials. Many buffer components, salts, and solubilizing detergents can interfere or prevent the successful application of both methodologies. Membranes, made of materials such as polyvinyldifluoride (PVDF), have been invaluable in this regard for protein sequencing. These membranes have allowed for direct analysis of proteins from complex mixtures by allowing for separation by gel electrophoresis and subsequent electroblotting that immobilizes the protein and removes potentially interfering small molecules (Matsudaira, 1987). PVDF membranes are also stable to most organic solvents. Mass spectrometry is a sensitive bioanalytical method (McCloskey, 1990), but it is often difficult for the method to selectively discriminate against most species found in a sample, in search of the few components the scientist is truly interested. MS analysis of a sample containing a small amount of peptide in a great molar excess of buffer salts typically results in a mass spectrum mostly composed of buffer ions. Common desalting or chromatographic methods are often necessary prior to analysis by mass spectrometry, but this adds an additional step of complexity and increases the total analysis time.

Keywords

Concentration Of20 Microchannel Plate Bovine Pancreatic Trypsin Inhibitor Electrospray Ionization Mass Spectrum Bovine Carbonic Anhydrase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Cody, R. B., Tamura, J., Finch, J. W., and Musselman, B. D., 1994, Improved detection limits for electrospray ionization on a magnetics sector mass spectrometer by using an array detector, J. Am. Soc. Mass Spectrom. 5: 194–200.CrossRefGoogle Scholar
  2. Cody, R. B., Tamura, J., and Musselman, B. D., 1992, Electrospray ionization/magnetic sector mass spectrome- try: calibration, resolution, and accurate mass measurements, Anal. Chem. 64: 1561–1570.CrossRefGoogle Scholar
  3. Feng, R., Bell, A., Dumas, F., and Konishi, Y., 1990, Reduction/alkylation plus ionspray mass spectrometry: a fast and simple method for accurate counting of cysteines, disulfide bridges and free SH groups in proteins, Proc. 38th ASMS Conf. on Mass Spectrom. Allied Topics, Tucson, AZ: American Society for Mass Spectrometry: East Lansing, MI, pp 273–274.Google Scholar
  4. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M., 1989, Electrospray ionization for mass spectrometry of large biomolecules, Science 246: 64–71.PubMedCrossRefGoogle Scholar
  5. Hillenkamp, F., Karas, M., Beavis, R. C., and Chait, B. T., 1991, Matrix-sssisted laser desorption/ionization mass spectrometry of biopolymers, Anal. Chem. 63: 1193A - 1203A.PubMedGoogle Scholar
  6. Karas, M., Bahr, U., Ingendoh, A., and Hillenkamp, F., 1989, Laser desorption/ionization mass spectrometry of proteins of mass 100 000 to 250 000 dalton, Angew. Chem. Int. Ed. Engl. 28: 760–761.CrossRefGoogle Scholar
  7. Loo, J. A., Edmonds, C. G., Udseth, H. R., and Smith, R. D., 1990, Effect of reducing disulfide-containing proteins on electrospray ionization mass spectra, Anal. Chem. 62: 693–698.PubMedCrossRefGoogle Scholar
  8. Loo, J. A., Ogorzalek Loo, R. R., and Andrews, P. C., 1993, Primary to quaternary protein structure determination with electrospray ionization and magnetic sector mass spectrometry, Org. Mass Spectrom. 28: 1640–1649.CrossRefGoogle Scholar
  9. Loo, J. A. and Pesch, R., 1994, Sensitive and selective determination of proteins with electrospray ionization magnetic sector mass spectrometry and array detection, Anal. Chem., 66: 3659–3663.CrossRefGoogle Scholar
  10. Matsudaira, P., 1987, Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes, J. Biol. Chem. 262: 10035–10038.PubMedGoogle Scholar
  11. McCloskey, J. A. (Ed.)., 1990, Mass Spectrometry. San Diego, CA: Academic Press.Google Scholar
  12. Smith, R. D., Loo, J. A., Edmonds, C. G., Barinaga, C. J., and Udseth, H. R., 1990, New developments in biochemical mass spectrometry: electrospray ionization, Anal. Chem. 62: 882–899.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Scott D. Buckel
    • 1
  • Tracy I. Stevenson
    • 1
  • Joseph A. Loo
    • 1
  1. 1.Division of Warner-Lambert CompanyParke-Davis Pharmaceutical ResearchAnn ArborUSA

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