Protein, you can’t hide anymore!

An improved method for the detection
of protein–protein interactions

A recent report published in the journal Molecular & Cellular Proteomics by scientists from The Rockefeller University details an improved method for detecting protein–protein interactions in a cell’s natural environment. In this method, dubbed stabilized affinity-capture mass spectrometry, or SAC-MS, the authors combined commonly used techniques and standardized a procedure to affinity capture proteins and subsequently identify them by mass spectrometry.

Protein–protein interactions are required in essentially all biological activities. A tremendous amount of research is being done to understand these interactions and their relationship to human diseases. A number of experimental techniques exist to isolate and identify these interactions, including isolation of protein complexes using specific antibodies (collectively termed affinity capture). Although traditional affinity-capture methods are used widely and have aided in scientific discoveries, they suffer from major shortcomings, such as inability to capture weak or transient protein interactors, and are unable to differentiate proximal (direct binding) from distant or indirect associations.

Brian Chait, who led the research project, describes this methodology as a “step forward in protein interaction studies in a cell’s native environment.” He added: “This technique allows us to capture and identify specifically associated proteins that may not have been feasible – such as transient and/or weak interactors, interactions that undergo rapid exchange, or even those that do not survive the normal biochemical isolation conditions used in conventional protein complex isolation methods.”

The SAC-MS technique has three steps:

  1. flash-freezing samples in liquid nitrogen to preserve the native cellular environment and molecular interactions, followed by fracturing samples at -80°C into microchunks;
  2. glutaraldehyde treatment to stabilize protein–protein interactions via crosslinking; and
  3. affinity-capture of associated proteins.
Affinity capture of proteinsThe stabilized affinity-capture mass spectrometry (SAC-MS) pipeline for targeted determination of specific protei–protein interactions and proximities in cellular milieu.

The affinity-captured complex then is subjected to mass spectroscopy to identify individual components.

The salient feature of this method, which sets it apart from other methods, is the use of sub-stoichiometric amounts of glutaraldehyde with respect to reactive amino acid residues in the protein. “This low molar ratio is optimized for stabilization of native interactions, efficient (affinity) isolation, and minimal interference with MS readout,” the authors wrote.

The authors validated the SAC-MS approach by studying two different protein complexes: the nuclear pore complex and the minichromosome maintenance complex. Their findings suggest that they have identified transient interactors, distinguished false positives from real interactors, and deciphered direct and indirect associations along with improved yields of many protein components of the complexes with SAC-MS.

Alok UpadhyayAlok Upadhyay ( alok7930@gmail.com) is a postdoctoral associate at Fox Chase Cancer Center. His major research area is Notch signaling regulation during cell fate decisions and neural crest stem cell development. Follow him on Twitter at www.twitter.com/alok7667.