NMR-based isotope editing, chemoselection and isotopomer distribution analysis in stable isotope resolved metabolomics

Abstract

NMR is a very powerful tool for identifying and quantifying compounds within complex mixtures without the need for individual standards or chromatographic separation. Stable Isotope Resolved Metabolomics (or SIRM) is an approach to following the fate of individual atoms from precursors through metabolic transformation, producing an atom-resolved metabolic fate map. However, extracts of cells or tissue give rise to very complex NMR spectra. While multidimensional NMR experiments may partially overcome the spectral overlap problem, additional tools may be needed to determine site-specific isotopomer distributions. NMR is especially powerful by virtue of its isotope editing capabilities using NMR active nuclei such as ¹³C, ¹⁵N, ¹⁹F and ³¹P to select molecules containing just these atoms in a complex mixture, and provide direct information about which atoms are present in identified compounds and their relative abundances. The isotope-editing capability of NMR can also be employed to select for those compounds that have been selectively derivatized with an NMR-active stable isotope at particular functional groups, leading to considerable spectral simplification. Here we review isotope analysis by NMR, and methods of chemoselection both for spectral simplification, and for enhanced isotopomer analysis.

Keywords: Chemoselection; Isotopomer distribution analysis; Stable isotope resolved metabolomics.

Figure 1.

Simulated and experimental splitting patterns in TOCSY and HCCH-TOCSY for a HCCH fragment A. TOCSY cross peak patterns expected for HCCH with different isotopomers as displayed on the figure. The area of each satellite is proportional to the fraction of each isotopomer present. Open circle is ¹²C, filled circles are ¹³C. [Adapted from Lane et al. (81) B. TOCSY spectrum showing ¹³C satellites at C5 and C6 in the uracil ring of UXP in an extract of LnCap cells grown in the presence of [U-¹³C]-glucose for 24 h. The pattern corresponds to a mixture of unlabeled ¹²C5¹²C6, ¹³C5¹²C6, ¹²C5¹³C6 and ¹³C5¹³C6 isotopomers. From Figure 4B of (82) licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), C. HCCH-TOCSY cross peak patterns expected for HC₁-C₂H-C₃H with different isotopomers as displayed on the figure. Cross-peaks are observed only when adjacent carbons are both ¹³C, i.e. have a significant ¹³C-¹³C coupling (typically 35–50 Hz). Open circles represent diagonal peaks, filled circles cross peaks.

Figure 1.

Simulated and experimental splitting patterns in TOCSY and HCCH-TOCSY for a HCCH fragment A. TOCSY cross peak patterns expected for HCCH with different isotopomers as displayed on the figure. The area of each satellite is proportional to the fraction of each isotopomer present. Open circle is ¹²C, filled circles are ¹³C. [Adapted from Lane et al. (81) B. TOCSY spectrum showing ¹³C satellites at C5 and C6 in the uracil ring of UXP in an extract of LnCap cells grown in the presence of [U-¹³C]-glucose for 24 h. The pattern corresponds to a mixture of unlabeled ¹²C5¹²C6, ¹³C5¹²C6, ¹²C5¹³C6 and ¹³C5¹³C6 isotopomers. From Figure 4B of (82) licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), C. HCCH-TOCSY cross peak patterns expected for HC₁-C₂H-C₃H with different isotopomers as displayed on the figure. Cross-peaks are observed only when adjacent carbons are both ¹³C, i.e. have a significant ¹³C-¹³C coupling (typically 35–50 Hz). Open circles represent diagonal peaks, filled circles cross peaks.

Figure 1.

Simulated and experimental splitting patterns in TOCSY and HCCH-TOCSY for a HCCH fragment A. TOCSY cross peak patterns expected for HCCH with different isotopomers as displayed on the figure. The area of each satellite is proportional to the fraction of each isotopomer present. Open circle is ¹²C, filled circles are ¹³C. [Adapted from Lane et al. (81) B. TOCSY spectrum showing ¹³C satellites at C5 and C6 in the uracil ring of UXP in an extract of LnCap cells grown in the presence of [U-¹³C]-glucose for 24 h. The pattern corresponds to a mixture of unlabeled ¹²C5¹²C6, ¹³C5¹²C6, ¹²C5¹³C6 and ¹³C5¹³C6 isotopomers. From Figure 4B of (82) licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), C. HCCH-TOCSY cross peak patterns expected for HC₁-C₂H-C₃H with different isotopomers as displayed on the figure. Cross-peaks are observed only when adjacent carbons are both ¹³C, i.e. have a significant ¹³C-¹³C coupling (typically 35–50 Hz). Open circles represent diagonal peaks, filled circles cross peaks.

Figure 2.. ¹³C coupling patterns in a 3-carbon fragment.

The expected splitting patterns in ¹³C NMR for a molecules such as alanine were calculated for ¹J_C1C2 = 52 Hz and ¹J_C2C3= 34 Hz. The small ²J_C1C3 ≈2 Hz was ignored. C1 was at f = 200 Hz, C2 at f=100 Hz and C3 at f=50 Hz. The line width was set to 2 Hz. A. Singlets corresponding to only one ¹³C at position 1 (red), 2 (blue) or 3 (green) B. Two ¹³C at C1C2, C2C3 and C1C3 C. All three ¹³C atoms simultaneously labeled D. Mixture of isotopomers. 25% ¹³C₁+ 25% ¹³C1¹³C2 +25% ¹³C2¹³C3 + 25% ¹³C1¹³C2¹³C3