ABRF-MIRG benchmark study: molecular interactions in a three-component system

J Biomol Tech. 2012 Sep;23(3):101-14. doi: 10.7171/jbt.12-2303-003.


Protein-protein interactions identified through high-throughput proteomics efforts continue to advance our understanding of the protein interactome. In addition to highly specific protein-protein interactions, it is becoming increasingly more common for yeast two-hybrid, pull-down assays, and other proteomics techniques to identify multiple protein ligands that bind to the same target protein. A resulting challenge is to accurately characterize the assembly of these multiprotein complexes and the competition among multiple protein ligands for a given target. The Association of Biomolecular Resource Facilities-Molecular Interactions Research Group recently conducted a benchmark study to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies, such as surface plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B, and C) and asked to determine if a ternary A-B-C complex can form or if protein-B and protein-C bind competitively to protein-A. This article will summarize the experimental approaches taken by participants to characterize the molecular interactions, the interpretation of the data, and the results obtained using different biosensor instruments.

Keywords: barnase; barstar; biolayer interferometry; biosensor; bivalent barnase variants; competition; epitope mapping; mass spectrometry; protein; proteomics; surface plasmon resonance; ternary complex.

MeSH terms

  • Bacterial Proteins / chemistry
  • Benchmarking*
  • Binding, Competitive
  • Humans
  • Immobilized Proteins / chemistry
  • Interferometry / standards
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping / standards*
  • Recombinant Fusion Proteins / chemistry
  • Reference Standards
  • Ribonucleases / antagonists & inhibitors
  • Ribonucleases / chemistry
  • Spectrometry, Mass, Electrospray Ionization / standards
  • Surface Plasmon Resonance / standards*


  • Bacterial Proteins
  • Immobilized Proteins
  • Recombinant Fusion Proteins
  • barstar protein, Bacillus amyloliquefaciens
  • Ribonucleases
  • Bacillus amyloliquefaciens ribonuclease