MS binding assays-with MALDI toward high throughput.
A novel type of MS binding assay, a substitute for radioligand binding, in which the quantification of the MS marker is performed by MALDI-MS-MS (FlashQuant) has been established. Because conventional MS binding assays can only be carried out by LC-ESI-MS-MS, the use of the FlashQuant system substantially increases the throughput capacity of this method. The study was performed for mGAT1 as a model system. First, a method was developed to quantify NO 711 as a marker for mGAT1 in a range from 208 pM to 16.7 nM using [2H10]NO 711 as an internal standard. On this basis, MS binding assays for mGAT1 could be implemented. Affinity constants determined in both saturation and competition experiments were in excellent agreement with those obtained in MS binding assays based on LC-ESI-MS-MS quantification. As the MALDI-MS system takes only a few seconds for quantification per sample, and the whole assay procedure is executed in a 96-well format, this technique is amenable to high-throughput screening.
Höfner
Georg
G
Department Pharmazie-Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 7, 81377 München, Germany.
Merkel
Dietrich
D
Wanner
Klaus Theodor
KT
D000818:Animals; D050485:GABA Plasma Membrane Transport Proteins / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D050485:GABA Plasma Membrane Transport Proteins