Quantifying Protein–Protein Interactions Within Noncovalent Complexes Using Electrospray Ionization Mass Spectrometry

Abstract

Several electrospray-mass spectrometry (ESI-MS)-based methods are available for determining the constant of association (K(a)) between a protein and a small ligand, but current MS-based strategies are not fully adequate for measuring K(a) of protein-protein interactions accurately. We expanded the application of ESI-MS-based titration to determine the strength of noncovalent interactions between proteins, forming a complex. Taking into account relative response factors (probability of being ionized, transmitted, and detected), we determined K(a) values of an equilibrium between dimers and tetramers at three different pH values (6.8, 3.4, and 8.4). We investigated the association of the lectin concanavalin A, whose dimer-tetramer ratio in the gas phase is affected by solution concentration and by pH. To calculate the constants of association in solution, we also utilized isothermal titration calorimetry (ITC) for a comparison with MS-based titration. At pH 6.8 and pH 8.4, the K(a) values measured by MS and by ITC were in agreement. ITC results allowed us to restrain the response factor to a value close to 4. At pH 3.4, we were able to measure the K(a) only by MS, but not by ITC because of limited sensitivity of calorimetry. Our investigation illustrates the great potential MS for calculating the binding strength of protein-protein interactions within noncovalent complexes. The main advantages of MS over ITC are its sensitivity (i.e., the required amount of sample is >100 times less than the one necessary for ITC), and the possibility to obtain precise information on composition of protein complexes, their stoichiometry, their subunit interactions, and their assembly pathway. Compared to previous investigations, our study shows the strong influence of response factors on determining accurate protein-protein association constants by MS.

Investigation of the chemical structure and formation mechanism of polydopamine from self‐assembly of dopamine by liquid chromatography/mass spectrometry coupled with isotope‐labelling techniques

“…The chromatographic peak at a migration time of 5.9 min containing the self‐assembled Dp oligomeric aggregates was also collected and analyzed via ESI‐Q‐TOF MS, a powerful analytical technique that has been used for a wide variety of applications . The analysis showed a strong [M + H] + ion signal for oxidized Dp at m/z 150.1 and signals from the corresponding [M + K] + adduct ion peak at m/z 188.0.…”

Extracting structural information from charge-state distributions of intrinsically disordered proteins by non-denaturing electrospray-ionization mass spectrometry

“…Evidence for that has been obtained by experimental and computational studies. Experimental evidence is based on direct investigation of the structural properties of gas-phase protein ions by ion mobility, 58 - 63 electron-capture dissociation, 64 , 65 gas-phase hydrogen exchange 66 , 67 and binding analysis 68 - 70 . Computational studies suggest that attractive interactions inside the protein structure can compensate to a certain extent for repulsive forces introduced by protein ionization 71 - 83 .…”

Mass Spectrometry-Guided Optimization and Characterization of a Biologically Active Transferrin–Lysozyme Model Drug Conjugate

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