Investigation of stable and transient protein-protein interactions: Past, present, and future

Abstract

This article presents an overview of the literature and a review of recent advances in the analysis of stable and transient protein-protein interactions (PPIs) with a focus on their function within cells, organs, and organisms. The significance of PTMs within the PPIs is also discussed. We focus on methods to study PPIs and methods of detecting PPIs, with particular emphasis on electrophoresis-based and MS-based investigation of PPIs, including specific examples. The validation of PPIs is emphasized and the limitations of the current methods for studying stable and transient PPIs are discussed. Perspectives regarding PPIs, with focus on bioinformatics and transient PPIs are also provided.

Figure 1. Roles of protein-protein interactions (PPIs) in organisms and methods for their investigation

(A) PPIs play an important role in every cellular process and thus in life as well. It is fair to say that there will be no life without the communication of proteins with each other. (B) Methods commonly used for the investigation of PPIs can be classified into four main groups: Biochemical, genetic, biophysical and microscopic. Of all these groups, biochemical techniques are the one most described in the literature and also most applied in experiments.

Figure 2. Identification of PPIs by BN-PAGE and LC-MS/MS (A) and by BN-PAGE and EM (B)

(A) The cell lysate of NG108 neuroblastoma x glioma cells was separated by BN-PAGE and then silver-stained. The gel bands corresponding to protein complexes were excised and digested by trypsin and further analyzed by LC-MS/MS. The MS/MS of peptides that were part of Eif3S10, Proteasome beta, Valosin-containing protein and ATP citrate lyase are shown. The sequences of the peptides are indicated for each spectrum. The approximate position in the gel for the proteins mentioned is shown. These proteins are parts of multisubunit-protein complexes (Eif3S10 is part of an 800 kDa complex and Proteasome beta is part of a 700 kDa complex) or homo-protein complexes (Valosin-containing protein is a 540 kDa homohexamer and ATP citrate lyase is a 480 kDa homotetramer). The Mw markers are indicated. (B) Isolated VE proteins were allowed to polymerize and then they were analyzed by SDS-PAGE (left), BN-PAGE (middle) and EM (right). Under denaturing and reducing conditions, VE proteins resolve in SDS-PAGE as monomeric proteins, but under native conditions, they polymerize into higher and higher polymeric structures. Polymerization pattern of VE gamma is different from the polymerization pattern of VE beta and each of them polymerizes different form the mixture of them. Magnification is x 15,000 (a), x 60,000 (b) and x 150,000 (c). (Figure adapted from reference with permission from the publisher.)

Figure 3. Investigation of PPIs by ESI-MS

ESI mass spectra of carbazole 1,9-dioxygenase (CarDO) (A) from a denaturing pH 3 solution containing 50% (v/v) acetonitrile showing the mass of the denatured 44.7 kDa polypeptide, and (B) from a 20 mM aqueous ammonium acetate, pH 6.8 solution showing the mass of the intact 134.8 kDa trimer complex (with each monomer bound to one 2Fe-2S cluster and one iron ion). The ESI mass spectrum of naphthalene dioxygenase (NDO) (C) from a pH 6.8 solution shows a mass of 218.6 kDa for an α₃β₃ complex with one 2Fe-2S cluster and one iron ion bound to each subunit. The inset figures show the zero charge deconvolution mass spectrum. (Figure adapted from reference with permission from the publisher.)

Figure 4. ESI-MS of native PPIs

Native ESI mass spectrum of intact HBV viral capsids from capsid particles reconstituted in vitro from truncated cp149 capsid monomers in 200 mM aqueous ammonium acetate, pH 6.8. The distribution of peaks around m/z 22,000 and 25,000 represent the T = 3 and T = 4 capsids, with a measured mass of 3,012 and 4,014 kDa, respectively. (Figure adapted from reference with permission from the publisher.)

Figure 5. Investigation of PPIs using DESI

Liquid sample DESI mass spectra of enolase (10 μM in water) with the DESI solvent composed of (A) 20 mM NH₄OAc (aq), (B) 1:1 ACN/H₂O with 1% FA, and (C) 1:1 ACN/H₂O with 1% FA and 40 mM m-NBA supercharging reagent. (Figure reprinted from reference with permission from the publisher.)