Quantitative chemical proteomics for identifying candidate drug targets

Anal Chem. 2003 May 1;75(9):2159-65. doi: 10.1021/ac026196y.

Abstract

We have developed a systematic strategy for drug target identification. This consists of the following sequential steps: (1) enrichment of total binding proteins using two differential affinity matrixes upon which are immobilized positive and negative chemical structures for drug activity, respectively; (2) covalent labeling of the proteins with a new cleavable isotope-coded affinity tag (ICAT) reagent, followed by proteolysis of the combined proteins; (3) isolation, identification, and relative quantification of the tagged peptides by liquid chromatography-mass spectrometry; (4) array-based transcription profiling to select candidate proteins; and (5) confirmation of direct interaction between the activity-associated structure and the selected proteins by using surface plasmon resonance. We present a typical application to identify the primary binding protein of a novel class of anticancer agents exemplified by E7070. Our results suggest that this approach provides a new aspect of quantitative proteomics to find specific binding proteins from protein mixture and should be applicable to a wide variety of biologically active small molecules with unidentified target proteins.

MeSH terms

  • Antineoplastic Agents / pharmacology
  • Chromatography, High Pressure Liquid
  • Drug Delivery Systems
  • Humans
  • Oligonucleotide Array Sequence Analysis
  • Proteomics / methods*
  • Sulfonamides / pharmacology
  • Surface Plasmon Resonance
  • Tetrazolium Salts
  • Thiazoles
  • Tumor Cells, Cultured

Substances

  • Antineoplastic Agents
  • N-(3-chloro-7-indolyl)-1,4-benzenedisulphonamide
  • Sulfonamides
  • Tetrazolium Salts
  • Thiazoles
  • thiazolyl blue