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Compound immobilization and drug-affinity chromatography.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2012; Vol. 803, pp. 25-38. - Publication Year :
- 2012
-
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.
- Subjects :
- Animals
Cell Extracts
Cells, Cultured
Chromatography, Liquid
Dasatinib
Electrophoresis, Polyacrylamide Gel
Immunoblotting
Mass Spectrometry
Proteins analysis
Pyrimidines chemistry
Pyrimidines pharmacology
Silver Staining
Thiazoles chemistry
Thiazoles pharmacology
Chromatography, Affinity methods
Drug Compounding methods
Pharmaceutical Preparations isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 803
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 22065216
- Full Text :
- https://doi.org/10.1007/978-1-61779-364-6_3