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Quantifying protein-protein interactions within noncovalent complexes using electrospray ionization mass

Elisabetta Boeri Erba¹, Konstantin Barylyuk, Yang Yang

¹Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.

Analytical Chemistry

|November 4, 2011

Summary

This study demonstrates electrospray-mass spectrometry (ESI-MS) can accurately measure protein-protein binding strength (K(a)). ESI-MS offers high sensitivity and detailed complex information, surpassing isothermal titration calorimetry (ITC) in certain applications.

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Area of Science:

Biochemistry
Analytical Chemistry
Mass Spectrometry

Background:

Electrospray-mass spectrometry (ESI-MS) methods for determining association constants (K(a)) are established for protein-ligand interactions.
Current ESI-MS strategies require refinement for accurate K(a) measurement of protein-protein interactions.

Purpose of the Study:

To adapt and validate ESI-MS titration for quantifying K(a) of protein-protein noncovalent interactions.
To compare ESI-MS K(a) measurements with isothermal titration calorimetry (ITC).

Main Methods:

Utilized ESI-MS-based titration to determine K(a) values for protein dimer-tetramer equilibria.
Accounted for relative response factors (ionization, transmission, detection probabilities).
Employed isothermal titration calorimetry (ITC) for comparative K(a) analysis.

Main Results:

ESI-MS accurately determined K(a) values for concanavalin A dimer-tetramer association at pH 6.8 and 8.4, agreeing with ITC.
ITC data helped refine the response factor for ESI-MS to approximately 4.
ESI-MS successfully measured K(a) at pH 3.4, where ITC lacked sensitivity.

Conclusions:

ESI-MS is a powerful tool for measuring protein-protein binding strengths in noncovalent complexes.
ESI-MS offers superior sensitivity and detailed complex composition insights compared to ITC.
Accurate K(a) determination by ESI-MS is strongly influenced by response factor considerations.