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Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization
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Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

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Using short columns to speed up LC-MS quantification in MS binding assays.

Georg Höfner1, Klaus Theodor Wanner

  • 1Department Pharmazie - Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr 7, 81377 München, Germany. ghoef@cup.uni-muenchen.de

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
|December 29, 2009
PubMed
Summary

Short columns significantly accelerate LC-MS quantification in MS binding assays, enhancing throughput without compromising accuracy. This method, exemplified by NO 711 binding to GAT1, offers rapid analysis for drug discovery.

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Area of Science:

  • Analytical Chemistry
  • Pharmacology
  • Biochemistry

Background:

  • Mass spectrometry (MS) binding assays offer an alternative to traditional radioligand binding by using MS for quantification.
  • High-throughput analysis is crucial for drug discovery and development.

Purpose of the Study:

  • To develop and validate rapid LC-MS quantification methods using short columns for MS binding assays.
  • To improve the throughput of MS binding assays for neurotransmitter transporter studies.

Main Methods:

  • Utilized short RP-18 columns (20 mm x 2 mm and 10 mm x 2 mm) with high flow rates (up to 1000 µL/min) in an isocratic mode.
  • Developed fast chromatography methods for NO 711 quantification using [(2H10)]NO 711 as an internal standard.
  • Validated methods according to FDA guidelines for linearity, accuracy, and precision.

Main Results:

  • Achieved short retention times (8-9s) and cycle times (18s) using short columns.
  • Enabled quantification of NO 711 from 50 pM to 5 nM directly from matrix samples.
  • Saturation experiment results (Kd- and Bmax-values) were comparable to those obtained with standard columns.

Conclusions:

  • Short columns are effective for speeding up LC-MS quantification in MS binding assays.
  • The developed methods are validated and suitable for quantifying NO 711 in saturation experiments.
  • This approach significantly enhances throughput for MS binding assays without sacrificing data quality.