Protein identification by solid phase microextraction—capillary zone electrophoresis—microelectrospray—tandem mass spectrometry

Abstract

We describe an analytical system for the rapid identification of proteins by correlation of tandem mass spectra with protein sequence databases. The system consists of an integrated solid phase microextraction/capillary zone electrophoresis peptide separation device that is connected through a microelectrospray Ion source to a tandem mass spectrometer. The limits of detection are 660 amol of sample at a concentration limit of <33 amol/μl for peptide mass measurement, and <10 fmol of sample, at a concentration limit of <300 amol/μl for peptide analysis by collision-induced dissociation. Using this system, we have identified low nanogram amounts of yeast proteins separated by high-resolution two-dimensional gel electrophoresis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Mann, M. and Wilm, M. 1994. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66: 4390–4399.

  2. 2

    Patterson, S.D. and Aebersold, R. 1995. Mass spectrometric approaches for the identification of gel-separated proteins. Electrophoresis 16: 1791–1814.

  3. 3

    Tomlinson, A.J., Braddock, W.D., Benson, L.M., Oda, R.P., and Naylor, S. 1995. Preliminary investigations of preconcentration-capillary electrophoresis-mass spectrometry. J. Chromatogr. B. 669: 65–73.

  4. 4

    Strausbauch, M.A., Landers, J.P., and Wettstein, P.J. 1996. Mechanism of pep-tide separations by solid phase extraction capillary electrophoresis at low pH. Anal. Chem. 68: 306–314.

  5. 5

    Strausbauch, M.A., Madden, B.J., Wettstein, P.J., and Landers, J.P. 1995. Sensitivity enhancement and second-dimensional information from solid phase extraction-capillary electrophoresis of entire high-performance liquid chromatog-raphy fractions. Electrophoresis 16: 541–548.

  6. 6

    Beattie, J.H., Self, R., and Richards, M.P. 1995. The use of solid phase concentrators for on-line preconcentration of metallothionein prior to isoform separation by capillary electrophoresis. Electrophoresis 16: 322–328.

  7. 7

    Eng, J., McCormack, A.L., and Yates, J.R.I. 1994. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5: 976–989.

  8. 8

    Yates, J.R.I., Eng, J.K., McCormack, A.L., and Schieltz, D. 1995. Method to correlate tandem mass-spectra of modified peptides to amino-acid-sequences in the protein database. Anal. Chem. 67: 1426–1436.

  9. 9

    Wahl, J.H. and Smith, R.D. 1994. Comparison of buffer systems and interface designs for capillary electrophoresis-mass spectrometry. J. of Capillary Electrophoresis 1: 62–71.

  10. 10

    Aebersold, R.H., Leavitt, J., Saavedra, R.A., Hood, L.E., and Kent, S.B. 1987. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc. Natl. Acad. Sci. USA 84: 6970–6974.

  11. 11

    Figeys, D., Ducret, A., Oostveen, I.V., and Aebersold, R. 1996. Protein identification by capillary zone electrophoresis-microelectrospray ionization-tandem mass spectrometry at the subfemtomol level. Anal. Chem. 68: 1822–1828.

  12. 12

    Andren, P.E., Emmett, M.R., and Caprioli, R.M. 1994. Zeptomole/attomole per microliter sensitivity for peptides. J. Am. Soc. Mass. Spectrom. 5: 867–869.

  13. 13

    Emmett, M.R. and Caprioli, R.M. 1994. Micro-electrospray mass spectrometry: Ultra-high-sensitivity analysis of peptides and proteins. J. Am. Soc. Mass. Spectrom. 5: 605–613.

  14. 14

    The National Center for Human Genome Research. 1996. International team completes DMA sequence of Yeast. Press Release,April 24th.

  15. 15

    Rabilloud, T. 1994. Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients. Electrophoresis 15: 278–282.

  16. 16

    Bleasby, A.J., and Wootton, J.C. 1990. Construction of validated, nonredundant composite protein sequence databases. Protein Engineering 3: 153–159.

  17. 17

    Boguski, M.S., Lowe, T.M.J., and Tolstoshev, C.M. stoshev, C. M. 1993. dbEST—database for “expressed sequence tags”. Nat. Genet. 4: 332–333.

  18. 18

    Boguski, M.S., Tolstoshev, C.M., and Bassett, D.E.J. 1994. Gene discovery in dbEST. Science 265: 1993–1994.

  19. 19

    Ducret, A., Bruun, C.F., Bures, E.J., Marhaug, G., Husby, G., and Aebersold, R. 1996. Characterization of human serum amyloid protein A isoforms separated by two-dimensional electrophoresis by liquid chromatography/tandem mass spectrometry. Electrophoresis 17: 866–876.

  20. 20

    Jazwinski, M. 1990. pp. 154–174 in Methods in enzymology “Guide to protein purification”. Deutscher, M.P. (ed.). Academic Press Inc., San Diego, CA.

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Figeys, D., Ducret, A., Yates, J. et al. Protein identification by solid phase microextraction—capillary zone electrophoresis—microelectrospray—tandem mass spectrometry. Nat Biotechnol 14, 1579–1583 (1996). https://doi.org/10.1038/nbt1196-1579

Download citation

Further reading