Skip to main content
SpringerLink
Log in

Expanding the scope of MS binding assays to low-affinity markers as exemplified for mGAT1

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Following a recently developed concept of MS binding assays based on the quantification of a native marker by LC–MS a procedure to study binding of a low-affinity marker in kinetic, saturation, and competition experiments was established. Separation of bound and unbound marker—the most crucial step of the assay—could be effectively achieved by filtration in a 96-well-format. MS binding assays according to this procedure allowed the reliable characterization of NO 711 binding to mGAT1 in presence of physiological NaCl concentrations. Comparing the results obtained in the present study with those from experiments using 1 mol L−1 NaCl in the incubation milieu reveals remarkable differences with respect to the marker’s affinity and kinetics and to the investigated test compound’s potency.

After incubation of a target with a native marker, bound and unbound marker are separated by filtration. Subsequently, the bound native marker is liberated from the target and finally quantified by LC-MS-MS.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Williams M, Mehlin C, Triggle DJ (2003) Receptor targets in drug discovery and development. In: Abraham DJ (ed) Burger’s medicinal chemistry and drug discovery, vol 2: Drug development. Wiley, New York

    Google Scholar 

  2. Höfliger MM, Beck-Sickinger AG (2003) Receptor–ligand interaction. In: Böhm HJ, Schneider G (eds) Protein–ligand interactions. From molecular recognition to drug design. Wiley–VCH, Weinheim

    Google Scholar 

  3. Lundqvist T (2005) Curr Opin Drug Discovery Dev 8:513–519

    CAS  Google Scholar 

  4. Cooper MA (2004) J Mol Recognit 17:286–315

    Article  CAS  Google Scholar 

  5. Siegel MM (2005) Mass-spectrometry-based drug screening assays for early phases in drug discovery. In: Lee MS (ed) Integrated strategies for drug discovery using mass spectrometry. Wiley, New York

    Google Scholar 

  6. Wanner KT, Höfner G (eds) (2007) Mass spectrometry in medicinal chemistry. Wiley–VCH, Weinheim

  7. Höfner G, Wanner KT (2003) Angew Chem Int Ed 42:5235–5237

    Article  Google Scholar 

  8. Höfner G, Wanner KT (2003) Angew Chem 115:5393–5395

    Article  Google Scholar 

  9. Suzdak PD, Frederiksen K, Andersen KE, Sorensen PO, Knutsen LJS, Nielsen EB (1992) Eur J Pharmacol 223:189–198

    Article  Google Scholar 

  10. Armer RE (2000) Curr Med Chem7:199–209

    CAS  Google Scholar 

  11. Braestrup C, Nielsen EB, Sonnewald U, Knutsen JS, Andersen KE, Jansen JA, Frederiksen K, Andersen PH, Mortnesen A, Suzdak PD (1990) J Neurochem 54:639–647

    Article  CAS  Google Scholar 

  12. Zepperitz C, Höfner G, Wanner KT (2006) Chem Med Chem 1:208–217

    Article  CAS  Google Scholar 

  13. Meldrum BS, Chapman AG (1999) Epilepsia 40:2–6

    Article  Google Scholar 

  14. Rovati GE (1998) Trends Pharmacol Sci 19:365–369

    Article  CAS  Google Scholar 

  15. Bylund DB, Murrin LC (2000) Life Sci 67:287–291

    Article  Google Scholar 

  16. Bennett JP Jr, Yamamura HI (1985) Neurotransmitter, hormone, or drug receptor binding methods. In: Yamamura HI, Enna SJ, Kuhar MJ (eds) Neurotransmitter receptor binding. Raven Press, New York

    Google Scholar 

  17. Hulme EC (1992) Centrifugation binding assays. In: Hulme EC (ed) Receptor–ligand interactions. A practical approach. IRL Press, Oxford

    Google Scholar 

  18. Cheng Y-C, Prusoff WH (1973) Biochem Pharmacol 22:3099–3108

    Article  CAS  Google Scholar 

  19. Kragler A, Höfner G, Wanner KT (2008) Eur J Med Chem, in print

  20. Bradford M (1976) Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. RCC Ltd, Zelgliweg 1, 4452, Itingen, Switzerland

    Christine Zepperitz

  2. Department für Pharmazie, Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5–13 Haus C, 81377, München, Germany

    Georg Höfner & Klaus T. Wanner

Authors
  1. Christine Zepperitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Georg Höfner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Klaus T. Wanner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Klaus T. Wanner.

Additional information

Dedicated to Prof. Hans-Dietrich Stachel on the occasion of his 80th birthday

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zepperitz, C., Höfner, G. & Wanner, K.T. Expanding the scope of MS binding assays to low-affinity markers as exemplified for mGAT1. Anal Bioanal Chem 391, 309–316 (2008). https://doi.org/10.1007/s00216-008-1956-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-008-1956-5

Keywords

Search

Navigation