Accurate quantification of protein concentrations and state populations by 2D ¹H-N Q-BEST NMR

We introduce 2D Q-BEST NMR as a quantitative approach for determining protein concentrations and relative state populations from a set of three ¹H-¹⁵N correlation spectra. The BEST methodology ensures short overall experimental times and highest sensitivity. The method was validated using ubiquitin as a model system, where a precision of approximately 15-20% was achieved at the level of individual cross-peaks. This precision can be further improved by averaging corrected intensities of well-resolved resonances corresponding to the same protein state. The approach was subsequently applied to the quantification of protein concentrations across multiple samples and to the determination of conformational state populations in proteins exhibiting intrinsic or ligand-induced heterogeneity. In both cases, 2D Q-BEST NMR provides accurate and robust quantification of NMR-visible species, demonstrating its utility as a general spectroscopic tool for quantitative biomolecular NMR analysis.