Skip to main content
SpringerLink
Log in

High-Sensitivity Peptide Mapping by Micro-LC with On-Line Membrane Blotting and Subsequent Detection by Scanning-IR-MALDI Mass Spectrometry

  • Published:
Journal of Protein Chemistry Aims and scope Submit manuscript

Abstract

A novel approach to the on-line mass determination of peptides from digested proteins by scanning infrared matrix-assisted laser desorption/ionization (scanning-IR-MALDI) is described. The peptides were continuously collected directly onto a PVDF (polyvinylidene fluoride) strip during a HPLC run. Individual peptides were detected by lining up the PVDF strip with the UV trace from the HPLC run, using visible dye markers as reference points. The local resolution of the peptides on the PVDF membrane is preserved during matrix incubation for MALDI-MS as shown by comparing the UV chromatogram and the total ion current (TIC) from an on-line coupled electrospray ionization (ESI) mass spectrometer with the scanning-IR-MALDI data from the corresponding areas on the PVDF strip. The intensities of the mass profiles obtained by scanning-IR-MALDI reflect the amount of peptides present on the PVDF strip. The higher sensitivity of IR-MALDI-MS yielded mass information not detectable by ESI-MS. After the scanning-IR-MALDI experiment, the same membrane strip can be used directly for automated Edman degradation. Comparable initial and repetitive yields were obtained for blotted peptides with and without matrix incubation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

REFERENCES

  • Blais, J. C., Nagnan-Le-Meillour, P., Bolbach, G., and Tabet, J. C. (1996). Rapid Commun. Mass Spectrom. 10, 1–4.

    Article  CAS  Google Scholar 

  • Covey, T. R., Bronner, R. F., Shushan, B. I., and Henion, J. (1988). Rapid Commun. Mass Spectrom. 2, 249–256.

    Article  CAS  PubMed  Google Scholar 

  • Eckerskorn, C., and Grimm, R. (1996). Electrophoresis 17, 899–906.

    Article  CAS  PubMed  Google Scholar 

  • Eckerskorn, C., Strupat, K., Karas, M., Hillenkamp, F., and Lottspeich, F. (1992). Electrophoresis 13, 664–665.

    Article  CAS  PubMed  Google Scholar 

  • Hsi, K.-L., Kochersperger, M. L., Werner, W. E., Grimley, C., Ly, H., Sandell, S., and Yuan, P. M. (1995). In PE Applied Biosystems: “Moving Forward in Protein Analysis” 1995, Posterior Presentations, pp. 1–10.

  • Mehl, E., Plambeck, J., and Lottspeich, F. (1993). In Würzburger Kolloquium 1993 (Engelhardt, H., ed.), Bertsch, Straubing, pp. 189–195.

    Google Scholar 

  • Schreiner, M., Strupat, K., Lottspeich, F., and Eckerskorn, C. (1996). Electrophoresis 17, 954–961.

    Article  CAS  PubMed  Google Scholar 

  • Strupat, K., Eckerskorn, C., Karas, M., and Hillenkamp, F. (1994a). In Proceedings of the 42nd ASMS Conference on Mass Spectrometry and Allied Topics.

  • Strupat, K., Karas, M., Hillenkamp, F., Eckerskorn, C., and Lottspeich, F. (1994b). Anal. Chem. 66, 464–470.

    Article  CAS  Google Scholar 

  • Vestling, M., and Fenselau, C. (1994). Anal. Chem. 64, 471–476.

    Article  Google Scholar 

  • Zhang, H., and Caprioli, R. M. (1996a). In Proceedings of the 44nd ASMS Conference on Mass Spectrometry and Allied Topics, THP237.

  • Zhang, H., and Caprioli, R. M. (1996b). J. Mass Spectrom. 31, 690–692.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Max-Planck-Institute for Biochemistry, 82152, Martinsried, Germany

    Christoph Eckerskorn, Kerstin Strupat, Josef Kellermann & Friedrich Lottspeich

  2. Institute for Medical Physics and Biophysics, University of Münster, 48149, Münster, Germany

    Christoph Eckerskorn & Franz Hillenkamp

  3. Institute for Medical Physics and Biophysics, University of Münster, 48149, Münster, Germany

    Kerstin Strupat

Authors
  1. Christoph Eckerskorn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kerstin Strupat
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Josef Kellermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Friedrich Lottspeich
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Franz Hillenkamp
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Eckerskorn, C., Strupat, K., Kellermann, J. et al. High-Sensitivity Peptide Mapping by Micro-LC with On-Line Membrane Blotting and Subsequent Detection by Scanning-IR-MALDI Mass Spectrometry. J Protein Chem 16, 349–362 (1997). https://doi.org/10.1023/A:1026324419398

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1026324419398

Search

Navigation