A suite of reduced-dimensionality ¹³ C, ¹⁵ N, ¹ H-triple-resonance NMR experiments is presented for rapid and complete protein resonance assignment. Even when using short measurement times, these experiments allow one to retain the high spectral resolution required for efficient automated analysis. “Sampling limited” and “sensitivity limited” data collection regimes are defined, respectively, depending on whether the sampling of the indirect dimensions or the sensitivity of a multidimensional NMR experiments per se determines the minimally required measurement time. We show that reduced-dimensionality NMR spectroscopy is a powerful approach to avoid the “sampling limited regime”—i.e., a standard set of ten experiments proposed here allows one to effectively adapt minimal measurement times to sensitivity requirements. This is of particular interest in view of the greatly increased sensitivity of NMR spectrometers equipped with cryogenic probes. As a step toward fully automated analysis, the program autoassign has been extended to provide sequential backbone and ¹³ C ^β resonance assignments from these reduced-dimensionality NMR data.

Stable isotope tracing with ultra-high resolution Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) can provide simultaneous determination of hundreds to thousands of metabolite isotopologue species without the need for chromatographic separation. Therefore, this experimental metabolomics methodology may allow the tracing of metabolic pathways starting from stable-isotope-enriched precursors, which can improve our mechanistic understanding of cellular metabolism. However, contributions to the observed intensities arising from the stable isotope's natural abundance must be subtracted (deisotoped) from the raw isotopologue peaks before interpretation. Previously posed deisotoping problems are sidestepped due to the isotopic resolution and identification of individual isotopologue peaks. This peak resolution and identification come from the very high mass resolution and accuracy of FT-ICR-MS and present an analytically solvable deisotoping problem, even in the context of stable-isotope enrichment.