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Analytical and Bioanalytical Chemistry

, Volume 391, Issue 1, pp 309–316 | Cite as

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

  • Christine Zepperitz
  • Georg Höfner
  • Klaus T. WannerEmail author
Original Paper

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.

Principle of MS binding assays

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.

Keywords

MS binding assays Mass spectrometry Ligand binding GAT1 GABA uptake LC–MS 

References

  1. 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 YorkGoogle Scholar
  2. 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, WeinheimGoogle Scholar
  3. 3.
    Lundqvist T (2005) Curr Opin Drug Discovery Dev 8:513–519Google Scholar
  4. 4.
    Cooper MA (2004) J Mol Recognit 17:286–315CrossRefGoogle Scholar
  5. 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 YorkGoogle Scholar
  6. 6.
    Wanner KT, Höfner G (eds) (2007) Mass spectrometry in medicinal chemistry. Wiley–VCH, WeinheimGoogle Scholar
  7. 7.
    Höfner G, Wanner KT (2003) Angew Chem Int Ed 42:5235–5237CrossRefGoogle Scholar
  8. 8.
    Höfner G, Wanner KT (2003) Angew Chem 115:5393–5395CrossRefGoogle Scholar
  9. 9.
    Suzdak PD, Frederiksen K, Andersen KE, Sorensen PO, Knutsen LJS, Nielsen EB (1992) Eur J Pharmacol 223:189–198CrossRefGoogle Scholar
  10. 10.
    Armer RE (2000) Curr Med Chem7:199–209Google Scholar
  11. 11.
    Braestrup C, Nielsen EB, Sonnewald U, Knutsen JS, Andersen KE, Jansen JA, Frederiksen K, Andersen PH, Mortnesen A, Suzdak PD (1990) J Neurochem 54:639–647CrossRefGoogle Scholar
  12. 12.
    Zepperitz C, Höfner G, Wanner KT (2006) Chem Med Chem 1:208–217CrossRefGoogle Scholar
  13. 13.
    Meldrum BS, Chapman AG (1999) Epilepsia 40:2–6CrossRefGoogle Scholar
  14. 14.
    Rovati GE (1998) Trends Pharmacol Sci 19:365–369CrossRefGoogle Scholar
  15. 15.
    Bylund DB, Murrin LC (2000) Life Sci 67:287–291CrossRefGoogle Scholar
  16. 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 YorkGoogle Scholar
  17. 17.
    Hulme EC (1992) Centrifugation binding assays. In: Hulme EC (ed) Receptor–ligand interactions. A practical approach. IRL Press, OxfordGoogle Scholar
  18. 18.
    Cheng Y-C, Prusoff WH (1973) Biochem Pharmacol 22:3099–3108CrossRefGoogle Scholar
  19. 19.
    Kragler A, Höfner G, Wanner KT (2008) Eur J Med Chem, in printGoogle Scholar
  20. 20.
    Bradford M (1976) Anal Biochem 72:248–254CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Christine Zepperitz
    • 1
  • Georg Höfner
    • 2
  • Klaus T. Wanner
    • 2
    Email author
  1. 1.RCC LtdItingenSwitzerland
  2. 2.Department für PharmazieZentrum für Pharmaforschung, Ludwig-Maximilians-Universität MünchenMünchenGermany

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