Compound immobilization and drug-affinity chromatography

Methods Mol Biol. 2012;803:25-38. doi: 10.1007/978-1-61779-364-6_3.

Abstract

Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Extracts
  • Cells, Cultured
  • Chromatography, Affinity / methods*
  • Chromatography, Liquid
  • Dasatinib
  • Drug Compounding / methods*
  • Electrophoresis, Polyacrylamide Gel
  • Immunoblotting
  • Mass Spectrometry
  • Pharmaceutical Preparations / isolation & purification*
  • Proteins / analysis
  • Pyrimidines / chemistry
  • Pyrimidines / pharmacology
  • Silver Staining
  • Thiazoles / chemistry
  • Thiazoles / pharmacology

Substances

  • Cell Extracts
  • Pharmaceutical Preparations
  • Proteins
  • Pyrimidines
  • Thiazoles
  • Dasatinib