Monitoring real-time metabolism of living cells by fast two-dimensional NMR spectroscopy.
Living cell metabolism is often monitored by 1D NMR spectroscopy, but the spectral resolution and the short cell lifetime are certainly limiting aspects. 2D spectroscopy does yield higher resolution but is time-consuming since acquisition of the second dimension requires several minutes. However, after only few minutes, oxygen starvation changes cell metabolism, and long acquisition times may yield spectra that do not represent the cell physiological state. Accordingly, metabolic studies of cells require fast NMR data acquisition. Here, we have applied band-selective optimized flip-angle short-transient (SOFAST)-HMQC techniques to (15)N-labeled cells, showing for the first time that it is possible to obtain 2D (1)H-(15)N correlation spectra of small metabolites directly in living cells, in a few seconds and with a high S/N ratio. SOFAST-HMQC spectra of (15)N-labeled Thalassiosira rotula diatoms cells can be acquired in 10-15 s, and, as an application, we have detected a progressive variation of the amino acid content when diatoms are exposed to UV-B radiation, with no need of long analytical procedures to quantify the metabolic changes. We believe that fast acquisition techniques can easily be extended to other cell systems, foreseeing a wide application in the emerging fields of metabolomics and metabonomics, being able to picture the "instantaneous" in-cell metabolism.
Motta
Andrea
A
Istituto di Chimica Biomolecolare del Consiglio Nazionale delle Ricerche, I-80078 Pozzuoli (NA), Italy. andrea.motta@icb.cnr.it
Paris
Debora
D
Melck
Dominique
D
D017377:Diatoms / Q000166:cytology* / Q000378:metabolism*; D055432:Metabolomics / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*