Continuous in vivo metabolism by NMR

Abstract

Dense time-series metabolomics data are essential for unraveling the underlying dynamic properties of metabolism. Here we extend high-resolution-magic angle spinning (HR-MAS) to enable continuous in vivo monitoring of metabolism by NMR (CIVM-NMR) and provide analysis tools for these data. First, we reproduced a result in human chronic lymphoid leukemia cells by using isotope-edited CIVM-NMR to rapidly and unambiguously demonstrate unidirectional flux in branched-chain amino acid metabolism. We then collected untargeted CIVM-NMR datasets for Neurospora crassa, a classic multicellular model organism, and uncovered dynamics between central carbon metabolism, amino acid metabolism, energy storage molecules, and lipid and cell wall precursors. Virtually no sample preparation was required to yield a dynamic metabolic fingerprint over hours to days at ~4-min temporal resolution with little noise. CIVM-NMR is simple and readily adapted to different types of cells and microorganisms, offering an experimental complement to kinetic models of metabolism for diverse biological systems. composition with 4-8 min resolution for chronic lymphoid leukemia. Sedimentation and line broadening are major factors that limit standard NMR measurements of complex samples like cells. Koczula et al. were able to mitigate sedimentation by immobilizing the single cells in agarose 13 .Alternatively, HR-MAS enables high-resolution NMR measurements on mixed-phase samples such as tissues 14 , or more recently, living organisms 10,15-18 with minimal line broadening. In this study, we extended HR-MAS to real-time continuous in vivo measurements of metabolism in cells. Using isotope editing, CIVM-NMR was able to reproduce and more directly observe a surprising branched-chain amino acid (BCAA) flux result reported last year in human myeloid leukemia cells 19 . We found that CIVM-NMR was not only easier but faster and more conclusive than traditional approaches for flux measurements in human cell cultures. We then applied CIVM-NMR to the multicellular filamentous fungus, N. crassa, in both aerobic and anaerobic environments. We observed highly reproducible dynamics in central carbon and amino acid metabolism with ~4 min resolution over 11 hours. The continuous nature of these measurements facilitated metabolite annotation, and semi-automated peak tracing provided relative quantification of known and unknown compounds. We developed several new MATLAB functions and workflows, freely available through GitHub, for the analysis and visualization of these novel data. As CIVM-NMR can be applied widely to cells, tissues, and small multicellular organisms, it enables new opportunities in fields such as developmental and chronobiology for monitoring high-resolution metabolic time-series data. Importantly, it will enable more robust and experimentally-based kinetic metabolic models for diverse biological systems.We thank the A. Simpson lab for discussions and advice on drilling holes in rotor caps, M. Case, Clifford Slayman, and Z. Lewis for discussions on Neuros...

Continuous in vivo Metabolism by NMR

“…B. Schuttler and L. Mao provided stimulating discussions on N. crassa modeling. This manuscript has been released as a Pre-Print (Judge et al, 2018) at https://www.biorxiv.org/content/early/2018/12/19/501577.…”

Metabolomics-driven strain improvement: A mini review

“…In the future, the effectiveness of metabolomics-driven strain improvement strategies may be elevated further through the advancements in different facets of metabolomics technology. For instance, more accurate reflection of the actual metabolic state of strains can be achieved through the recent advancements in single-cell metabolomics (Hu et al, 2021) and real-time analysis (Judge et al, 2019). Spatial information of metabolite distribution can also be obtained through the combination of mass spectrometry and imaging techniques (Schleyer et al, 2019;Taylor et al, 2021).…”

Quantitative Isotopomer Rates in Real‐Time Metabolism of Cells Determined by NMR Methods

“…The two most common analytical technologies in this context are mass spectrometry (MS) [1] and Nuclear Magnetic Resonance (NMR) spectroscopy. NMR is becoming an increasingly important method in this context [2] as it has the ability to quantify site-specific label incorporation and is suitable for detecting metabolism in primary patient cells placed in an NMR tube t measure metabolism in real-time [2][3][4] and to analyze labelled cell extracts in reasonable highthroughput [5][6][7][8][9][10] . NMR offers several options to measure 13 C or 15 N isotope incorporation.…”