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. 2019 Feb 21;9:2520. doi: 10.1038/s41598-018-37525-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Sections from ¹H-¹³C HSQC spectra of polar cell extracts illustrating spectral improvements obtained using acquisition mode of the indirect directions States rather than Echo-AntiEcho in HSQCs with NUS. (a–d) Show slices and spectral regions for L-lactic acid resonances from ¹H,¹³C-HSQC spectra for HuH7 cells grown for 12 hours in a medium containing [U-¹³C] glucose. Echo-antiecho-¹H,¹³C-HSQC spectra acquired for samples in a mixed 90% H₂O/10% D₂O NMR buffer (panels a and b) and conventional ¹H,¹³C-HSQC spectra acquired for samples in a D₂O buffer (panels c and d). The high resolution in the ¹³C dimension obtained with 25% NUS sampling over 8k complex points is illustrated in panels e and f where the different couplings obtained from the PC-derived ¹³C₂–¹³C₃ and PDH-derived ¹³C₂-¹³C₁ moieties are clearly resolved in the L-aspartic acid (e) and L-malic acid (f) H₂C₂ resonances. (g) shows the alanine H2/C2 resonance at 3.78/53.2ppm from GLOR cells labelled with [U-¹³C]glucose and [¹⁵N]glutamine. The complexity of the ¹⁵N-undecoupled (blue) spectrum arises from the superimposition of resonances from mainly [¹³C-]alanine with a small proportion of [¹⁵N,¹³C]alanine. While J_C2C3 and J_C1C2 produce a well-resolved doublet of doublets. For [¹⁵N,¹³C]alanine, these 4 resonances are further split by the small J_C2N2 coupling. The resulting spectrum is further complicated by a ¹⁴N/¹⁵N isotope shift that causes overlap of one of the double components of [¹³C,¹⁵N]alanine with the resonance from [¹³C]alanine. (f): AXP (ADP and AMP) H₁/C₁ resonances at 6.15/89.7ppm from [¹⁵N,¹³C]glucose and [¹⁵N]glutamine labelled GLOR cells showing a complex pattern based on a doublet due to the large ¹J_C1C2 coupling in the undecoupled (blue) spectrum. Additional resonances are seen because a proportion of the C1 is directly attached to a ¹⁵N in the adenine ring. This complexity is reduced by ¹⁵N decoupling in the red spectrum but the remaining peaks appear broadened by the ¹⁴N/¹⁵N isotope shift⁴⁵ on the directly-attached ¹³C.