Quantitative affinity purification mass spectrometry: a versatile technology to study protein-protein interactions

Abstract

While the genomic revolution has dramatically accelerated the discovery of disease-associated genes, the functional characterization of the corresponding proteins lags behind. Most proteins fulfill their tasks in complexes with other proteins, and analysis of protein-protein interactions (PPIs) can therefore provide insights into protein function. Several methods can be used to generate large-scale protein interaction networks. However, most of these approaches are not quantitative and therefore cannot reveal how perturbations affect the network. Here, we illustrate how a clever combination of quantitative mass spectrometry with different biochemical methods provides a rich toolkit to study different aspects of PPIs including topology, subunit stoichiometry, and dynamic behavior.

Keywords: cross-linking; mass spectrometry based proteomics; protein–protein interaction; quantitative proteomics; stoichiometry.

FIGURE 1

Approaches to q-AP-MS experiments. (A) The left hand side depicts the typical workflow of a SILAC-based q-AP-MS experiment. Differentially SILAC-labeled cells are transfected with a tagged protein of interest or a control vector containing only the tag, respectively. Proteins are immunoprecipitated with antibodies directed against the tag. Samples are mixed prior to elution. Eluted proteins are cleaved into peptides and analyzed by Liquid-Chromatography Mass Spectrometry (LC-MS). (B–G) The right hand side depicts how q-AP-MS can be employed to study different aspects of PPIs. (B) Label-free quantification provides an alternative to SILAC. (C) Immunoprecipitation can compare changes in PPIs upon perturbation. (D) Transient interactions and complex structure can be studied by cross-linking. (E) Submodule composition and PPI dynamics can be revealed by sequential elution with increasing concentrations of SDS. (F) Limited proteolysis provides a means to detect interaction interfaces. (G) The stoichiometry of complexes can be revealed by comparing abundances of the different subunits.