Mass spectrometry assisted assignment of NMR resonances in reductively 13C-methylated proteins

Reductive 13C-methylation of proteins has been used as an isotope labeling strategy to study protein structure, function, and dynamics by nuclear magnetic resonance (NMR) spectroscopy. However, assigning the resulting 13C-dimethylamine peaks in a 1H-13C NMR spectrum has proved to be difficult, but it is important to expand the scope of the method. The assignment strategy presented here utilizes mass spectrometry (MS) for sequence identification and varying 13C/12C isotope ratios to correlate with NMR data. The site-specific reactivity of the lysines and N-terminal amine of a protein is exploited to produce a sample with varying 13C/12C ratios at each dimethylamine. MS and NMR are used to quantitate and correlate these ratios in order to assign peaks in the 1H-13C NMR spectrum. Hen egg white lysozyme was used as a model protein to demonstrate this assignment strategy.