Identification of protein interactions involved in cellular signaling

Abstract

Protein-protein interactions drive biological processes. They are critical for all intra- and extracellular functions, and the technologies to analyze them are widely applied throughout the various fields of biological sciences. This study takes an in-depth view of some common principles of cellular regulation and provides a detailed account of approaches required to comprehensively map signaling protein-protein interactions in any particular cellular system or condition. We provide a critical review of the benefits and disadvantages of the yeast two-hybrid method and affinity purification coupled with mass spectrometric procedures for identification of signaling protein-protein interactions. In particular, we emphasize the quantitative and qualitative differences between tandem affinity and one-step purification (such as FLAG and Strep tag) methods. Although applicable to all types of interaction studies, a special section is devoted in this review to aspects that should be considered when attempting to identify signaling protein interactions that often are transient and weak by nature. Finally, we discuss shotgun and quantitative information that can be gleaned by MS-coupled methods for analysis of multiprotein complexes.

Fig. 1.

Protein interactions mediate cellular responses. A hypothetical signal transduction pathway where sequential and transient interactions between upstream and downstream components of this pathway are active. All depicted interactions both between signaling proteins and with other cellular constituents are critical for proper signaling. They also are potential targets for disease causing alterations as well as for therapeutic targeting.

Fig. 2.

Dynamic changes in protein expression revealed through the use of metabolic labeling using stable isotope. A, cells are first labeled with amino acid isotopes and subsequently biologically stimulated. B, cells are lysed at various time points following stimulation and harvested, and proteins are purified, and peptides are generated. Subsequently, tryptic peptide precursor ion measurements are recorded by MS. C, following normalization and plotting, specific protein expression changes are recorded as a function of time following stimulation.