Authors
Aaron P. Yamniuk1, Suzanne C. Edavettal1, Simon Bergqvist2, Satya P. Yadav3, Michael L. Doyle1, Kelly Calabrese4, James F. Parsons4 and Edward Eisenstein4
Organizations
- Bristol-Myers Squibb, Princeton, New Jersey 08540, USA
- Pfizer, La Jolla, California 92121, USA
- Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA;
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville Maryland 20850, USA
Abstract
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.
CovalX Technology Used (Click each option to learn more)
Outcomes
This article summarizes the experimental approaches taken by 12 biosensor participants to accurately characterize the assembly of three unknown multiprotein complexes and the competition among multiple protein ligands for a given target protein. The participants were given microgram quantities of three proteins (A (barstar), B (barnase), and C (BiNase2)), 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.
One of the 12 participants (Participant 31) used the Mass Spectrometry (MS) approach where a solution made of various complexes was formed followed by chemical cross-linking and detection of the high molecular weight complexes using MALDI-TOF MS equipped with a CovalX HM2 high-mass detector. During the observation of A:C:A complex, it was found that barstar binding to the high- and low-affinity domains of BiNase2 was the result of higher protein concentrations. Due to lower concentrations used in biosensor experiments, this interaction was only detected by this participant. Participant 31 also confirmed the identity of the A:C:A complex in an independent experiment by mixing protein-C with excess protein-A
The results from the participants demonstrated the flexibility of today’s biosensor platforms in allowing numerous variations on common experimental designs. The MS-based technologies highlighted the continued emergence of these label-free techniques as complementary tools in the characterization of protein interactions.