Knocking Out False Positives In Interaction Proteomics

Matthias Mann and Matthias Selbach used antibody 'baits' immobilized on solid supports to fish out a target protein. Proteins that bind to the target protein under normal cellular conditions are thus also isolated along with the target protein itself. However, other proteins can cross-react with or nonspecifically bind to the antibody bait or solid support. These nonspecific interactions are also detected by the readout method and are known as false positives.

To control for this problem, the authors used a technique called RNA interference to knock out, or turn off, expression of the target protein in one population of cells. If the target protein is absent, only the nonspecific and cross-reactive binders will interact with the antibody bait. All of the isolated proteins from both the normal cell population and the knocked-out population are then identified by mass spectrometry. Proteins that are present in both cell populations are thus identified as false positives and discounted. This method is likely to become an important proteomic tool for studying human proteins, given the diverse range of antibodies and RNA interference reagents currently available.

Author contact:

Matthias Mann (Max Planck Institute of Biochemistry, Martinsreid, Germany)
E-mail: mmann@biochem.mpg.de

Abstract available online.

(C) Nature Methods press release.

Message posted by: Trevor M. D'Souza