J -resolved 1 H NMR 1D-projections for large-scale metabolic phenotyping studies: Application to blood plasma analysis Andrea Rodriguez-Martinez † , Joram M. Posma † , Rafael Ayala † , Nikita Harvey † , Beatriz Jimenez † , Ana L. Neves † , John C. Lindon † , Kazuhiro Sonomura ‡§ , Taka-Aki Sato ‡§ , Fumihiko Matsuda § , Pierre Zal- loua ║ , Dominique Gauguier †θ , Jeremy K. Nicholson †* , Marc-Emmanuel Dumas †* † Computational and Systems Medicine, Department of Surgery and Cancer, Imperial College London, London, UK. ‡ Life Science Research Center, Technology Research Laboratory, Shimadzu Corporation, Kyoto, Japan. § Center for Genomic Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan. ║ School of Medicine, Lebanese American University, Beirut, Lebanon. θ Cordeliers Research Centre, INSERM UMR_S 1138, Paris, France. ABSTRACT: 1 H nuclear magnetic resonance (NMR) spectroscopy-based metabolic phenotyping is now widely used for large- scale epidemiological applications. In order to minimize signal overlap present in 1D 1 H NMR spectra, we have investigated the use of 2D J-resolved (JRES) 1 H NMR spectroscopy for large-scale phenotyping studies. In particular, we have evaluated the use of the 1D projections of the 2D JRES spectra (pJRES), which provide single peaks for each of the J-coupled multiplets, using 705 human plasma samples from the FGENTCARD cohort. Based on the assessment of several objective analytical criteria (spectral disper- sion, attenuation of macromolecular signals, cross-spectral correlation with GC-MS metabolites, analytical reproducibility and biomarker discovery potential), we concluded that the pJRES approach exhibits suitable properties for implementation in large- scale molecular epidemiology workflows. In the last two decades, metabolic phenotyping (metabotyp- ing 1 ) has become a versatile approach for studying metabo- lism, with a broad range of applications in the fields of clinical biochemistry, toxicology and drug metabolism 2-4 . The main analytical techniques used for metabolic profiling are proton nuclear magnetic resonance ( 1 H NMR) spectroscopy and mass spectrometry (MS) coupled to liquid chromatography (LC- MS) or gas chromatography (GC-MS). NMR spectroscopy is a quantitative, non-destructive and highly reproducible tech- nique that provides detailed information of molecular struc- tures based on atom-centered nuclear interactions and proper- ties 5,6 . These qualities have made 1 H NMR spectroscopy one of the key analytical strategies for metabotyping in large-scale epidemiological studies 2,7-9 . The vast majority of 1 H NMR-based metabolic phenotyping studies employ one-dimensional (1D) experiments, as they allow relatively rapid spectral acquisition and therefore max- imize the throughput. However, 1D NMR spectroscopy leads to complex mixture spectra, with considerable peak overlap, thus limiting the number of metabolites that can be unambigu- ously identified and quantified 10 . One possibility to reduce the burden of spectral overlap and increase metabolite specificity is to resolve the multiplet resonances into a second dimension (i.e. 2D-NMR). J-resolved (JRES) 1 H NMR spectroscopy 11 is a very useful 2D 1 H NMR method for structural assignments because it efficiently separates chemical shift (δ) in the F2 dimension from J-coupling in the F1 dimension, and data acquisition times are amenable to be comparable to those used for other 1D 1 H NMR experiments 12 . The JRES experiment is composed of an array of spin-echo pulses, where an incremented delay period is applied to define the second dimension. In a JRES spectrum, both chemical shifts and homonuclear J-couplings appear in F2 and only homonuclear couplings appear in F1 (heteronuclear coupled multiplets behave like chemical shifts). The J-couplings and chemical shifts are resolved into two orthogonal axes (F1 and F2, respectively) by tilting the spectrum through 45° and also by symmetrizing the display to choose the lowest intensity of the two data points equidistant from the F1=0 axis 13 . The resonance intensities are edited according to proton T 2 relaxa- tion times 14 . The sum or skyline projection of a 2D JRES spectrum along the chemical shift axis effectively yields a virtual 1 H broadband proton-decoupled spectrum (pJRES) with interesting properties in terms of signal decomposition: i) there are fewer resonances in the pJRES spectrum than in a classical 1D spectrum for the same number of metabolites; and ii) multiplets have a smaller footprint along the chemical shift axis. Additional advantages of the JRES experiment over several other homonuclear decoupling (i.e. “pure shift”) meth- ods 15,16 are the efficient attenuation of broad resonances from macromolecules and the provision of J-coupling data, which can aid metabolite assignment 14,17 . JRES spectroscopy is par- ticularly suitable for metabotyping because the majority of endogenous metabolites of interest exhibit first-order spin systems, and consequently artifactual peaks associated with second-order spin systems are rare, especially at high magnet- ic fields 14 . In the last three decades, the advantages of JRES- based NMR spectroscopy have been reflected in several stud- ies demonstrating the potential of the JRES approach for