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PMID:28272839Spectra were acquired on a Bruker Avance III 600 MHz NMR spectrometer with a 1.7 mm CPTCI z-PFG cryogenic probe at 300 K. The 1H carrier was on the water frequency and the 1H 90° pulse was calibrated at a power of 0.326 W.University of BirminghamKaren AtkinsmixtureMCF-7 cellNMR spectroscopy6 plates incubated under the same condition were combined together for each replicate. Plates were processed in the following way. 1 ml of cold methanol (100% v/v) was added to one plate. 1 min later 1 ml of cold water was added to the same plate. The content of the plate was scraped and all the content was transferred to a second plate. The content of this plate was scraped and was transferred to a third plate. This process was followed with the 6 plates of the same replicate. After scraping the 6th plate, all the content was transferred to a glass tube, where 2 ml of cold chloroform was added. Tubes were vigorously agitated for 30 min at 4 ºC. After agitation, 1/6 part of the content of the tube was transferred to a separate tube. The tube containing the 1/6 part was used for GC/MS analysis and the tube containing the 5/6 part was used for NMR analysis. Next, tubes were centrifuged and the upper phase containing the polar metabolites was transferred to a separate glass tube. The tubes containing the polar phase for NMR analysis were frozen at -80 °C until their analysis.reference compoundHomo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS241Anusha Jayaraman. Universitat de Barcelona. Av. Diagonal 643 Edifici Prevosti floor -2. 08028 Barcelona (Spain). anusha.jayaraman@ub.edu. +34-934 021 217.Silvia Marin. Universitat de Barcelona. Av. Diagonal 643 Edifici Prevosti floor -2. 08028 Barcelona (Spain). silviamarin@ub.edu. +34-934 021 217.Christian Ludwig. Institute of Metabolism and Systems Research. College of Medical and Dental Sciences University of Birmingham Edgbaston Birmingham B15 2TT UK. C.Ludwig@bham.ac.uk. 0121 414 8362.Marta Cascante. Universitat de Barcelona. Av. Diagonal 643 Edifici Prevosti floor -2. 08028 Barcelona (Spain). martacascante@ub.edu. +34-934 021 593.Ulrich Gunther. School of Cancer Sciences. College of Medical and Dental Sciences University of Birmingham Edgbaston Birmingham B15 2TT UK. u.l.gunther@bham.ac.uk. 0121 414 8361.Daniel Tennant. Institute of Metabolism and Systems Research. College of Medical and Dental Sciences University of Birmingham Edgbaston Birmingham B15 2TT UK. D.Tennant@bham.ac.uk. (0121) 4148651.Pedro de Atauri. Universitat de Barcelona. Av. Diagonal 643 Edifici Prevosti floor -2. 08028 Barcelona (Spain). pde_atauri@ub.edu. +34-934 021 217.Mei Chong. School of Cancer Sciences. College of Medical and Dental Sciences University of Birmingham Edgbaston Birmingham B15 2TT UK. mei.chong@kcl.ac.uk. 0121 414 8368.1D 1H-NMR spectra were apodised using 0.3 Hz line broadening before zero-filling the fid data to 131072 data points prior to Fourier transformation. 2D 13C-HSQC spectra were reconstructed via the compressed sensing algorithm using the MDDNMR and NMRpipe software. The final data size was 1024 x 16384 real data points in the spectra. A 90° phase shifted sine squared was used to apodize the data in both dimensions. All NMR spectra were manually phase corrected. The 1D NMR spectra were baseline corrected using a spline function.tracerk.l.atkins@bham.ac.ukMCF-7 cells were seeded (1,000,000 per p100 plate) and 6 ml of medium was used in each of them. In total 48 plates were seeded - 6 plates combined together for each replicate (as 1:5 ratio for GCMS:NMR). The medium used was basal medium DMEM (A14430, Invitrogen) with 10% dialyzed FBS (F0392, Sigma), 0.01 mg/ml Insulin (19278, Sigma), 1% Non-Essential Amino acids (01-340-1B, Biological Industries), 10 mM glucose (G8769, Sigma) and 4 mM Glutamine (25030-81, Life Technologies). After 48 h of cell seeding, media was removed and plates were washed twice with PBS. 6 ml of fresh media containing tracers were added to each plate. The conditions used in this experiment were cold natural samples with [U-12C]-glucose (unlabeled glucose) (A), samples with tracer [U-13C6]-glucose (B) and samples with tracer [1,2-13C2]-glucose (C), amounting to 10 mM of glucose in each case. Time points of 0 h (before adding tracer) and 8 h (after tracers incubation) were maintained. 3 replicates for each condition at 8 h of tracers incubation (B and C) and 1 replicate for cold natural (A) at 8 h were obtained. In addition 3 plates were used for 0 h cell counting and another 3 for 8 h cell counting. After 8 h incubation, medium was removed and kept for biochemical concentration analysis. Plates were washed twice with cold PBS followed by snap-freezing using liquid nitrogen. Plates were kept under -80 ºC until their analysis. The commercial glucose standard mixture used for validation consisted of 10 mM with the composition of [U-12C]-D-glucose (82%) + [1,2-13C]-D-glucose (10%) + [1-13C]-D-glucose (3%) + [1,6-13C]-D-glucose (5%).1D-NOSEY: The standard Bruker pulse sequence noesygppr1d was used for a 1D NOESY with water pre-saturation. Key parameters were as follows: spectral width 12.15ppm/7288.6Hz; complex points, TD 32768; interscan delay, d1 4s; acquisition time, aq 4.50s ; short NOE mixing time, d8 10ms; number of scans, ns=128; dummy scans, ds=416. Custom filtered 1D-NOESY: Two spectra were acquired per sample using a yet unpublished quantitative filter pulse sequence, where one spectrum contains signals from all protons and the second spectrum only signals from protons directly attached to a 13C nucleus. NOESY pre-saturation was used for water suppression with a 10ms NOESY mixing time. Key parameters were as follows: spectral width 12.15ppm/7288.6Hz; complex points, TD 4096; interscan delay, d1 4s; acquisition time, aq 0.320s; number of scans, ns=128; dummy scans, ds=16. 2D-HSQC: For the 13C-1H HSQCs, the pulse sequence used was based on the Bruker standard pulse program hsqcetgpsp which uses Echo/Antiecho-TPPI gradient selection, with additional gradient pulses to improve water suppression. Key parameters for the 1H observe dimension were: spectral width, 7812.5Hz/13.01ppm; complex points, TD 1024. For the 13C indirect dimension, the key parameters were as follows: the 13C carrier was set to 80ppm; complex points, 30% out of 16384 complex points (TD 4096) were sampled using exponentially weighted non-uniform sampling; spectral width 24 kHz/160.0ppm. With dummy scans, ds 8; number of scans per increment, ns 2; interscan delay, d1 1.5s; acquisition time, aq 0.32s.MetabolomicsisotopomerFluxomicstracer-based metabolomicsnuclear magnetic resonance spectroscopybreast cancerisotopomerFluxomicstracer-based metabolomicsnuclear magnetic resonance spectroscopybreast cancerFor NMR analysis, extracts were reconstituted in 100mM sodium phosphate buffer (pH 7.0) containing 500µM TMSP (sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4), 1.5 mM sodium azide and 10% D2O and transferred to a 1.7 mm tube in a Bruker SampleJet autosampler.Cellular metabolism in mammalian cells represents a challenge for analytical chemistry in the context of current biomedical research. Mass spectrometry and NMR spectroscopy together with computational tools have been used to study metabolism in cells. Compartmentalization of metabolism complicates the interpretation of stable isotope patterns in mammalian cells owing to the superimposition of different pathways contributing to the same pool of analytes. This indicates a need for a model-free approach to interpret such data. Mass spectrometry and NMR spectroscopy provide complementary analytical information on metabolites. Herein an approach that simulates 13 C multiplets in NMR spectra and utilizes mass increments to obtain long-range information is presented. The combined information is then utilized to derive isotopomer distributions. This is a first rigorous analytical and computational approach for a model-free analysis of metabolic data applicable to mammalian cells.Combined Analysis of NMR and MS Spectra (CANMS).Chong Mei M, Jayaraman Anusha A, Marin Silvia S, Selivanov Vitaly V, de Atauri Carulla Pedro R PR, Tennant Daniel A DA, Cascante Marta M, Günther Ulrich L UL, Ludwig Christian Cchemical analysis., assay, determinationl(3)rK137, IPP2A2, determination, Glukose, Metabonomic, Monohydrate, Metabonomics, Measure, (DL)-Isomer, CG17228, Cost-Minimization, 1135/09, 5730420M11Rik, 1135/07, Techniques, Method, Dextrose, 0451/09, Software Engineering, Analysis, Cost Comparison, breast carcinoma cell line, Computer Program, SET, 0244/09, me75, Divorced, Analyses, TAF-I, developmental field, Cost-Minimization Analyses, N, Open, DROPROSA, Computer Programs and Programming, Comparison, Divorces, procedures, l(3)j6E2, D17Mit170, T1, DL-glucose, MCF7 cell, DmelCG4063, DmelCG4299, 671/2, allergic reaction, Cost Analysis, glucose, IGAAD, set, 0320/10, DMPROSPER, Methodological Studies, DmelCG10574, label., scientific observation, sample, Tbl1, TBL1, Comparisons, Affordabilities, PIK3CA-Related Overgrowth Spectrum, phapii, THPH5, l(3)rO534, THPH6, Pro, Krebs cycle, incomplete, StF-IT-1, future organ, Procedure, Tl3, Tl2, Source Softwares, PSA, Software Tools, Programs, abolished, Program, Computer Applications, PROS-1, PROS-2, Stickstoff, pro, 1316/02, Computer Applications Software, Computer Applications Softwares, BcDNA:HL08040, Softwares, (alpha-D)-Isomer, finances, D-Glucose, Software Applications, HLA-DR-associated protein II, CG4063, Voila, DI-2, Source Software, I-2Dm, 0664/07, 1167/13, salaries, CG4299, Methodological, Methodological Study, E-2f, E-2g, experimental procedures, I-2PP1, MCF7, Applications, nitrogen, TAF-IBETA, D Glucose, label, TAF-Ibeta, financial management, 7N, 0989/01, Glucose Monohydrate, i2pp2a, Cost Analyses, Computer Software Applications, gluco-hexose, Procedures, Tb11, experimental, Glucose, Cost Comparisons, Computer, 0763/13, PHAPII, l(3)10419, FBXW4, organ field, DmelCG17228, Cost Minimization Analysis, sensitive, Studies, anon-WO0140519.15, citric acid cycle, field, Low, Prosp, Metabolomic, Separated, sensitivity, Technique, Application, study, Open Source Softwares, PROS, 0585/13, anatomical systems, methods, Cost Measures, experimental section, Software Application, Ebi, EBI, Affordability, ipp2a2, Open Source Software, Measures, 2pp2a, labeling, financing, CG10574, Computer Software Application, l(3)rH013, Study, funding, 2PP2A, Tools, taf-ibeta, dSET, dSet, liquid, 0563/18, Anhydrous, MCF-7 cell, 0671/02, Applications Software, l(3)j12C8, fees, measuring, Open Source, cou, Computer Software, (beta-D)-Isomer, igaad, SMAP55, Cell, l(3)rJ806, group, Tool, PS24, Pros, PS23, PS25, l(3)rL433, Software Tool, PS22, Lr, Experiment, PS21, l(3)rI160, 0441/16, I-2PP2A, oxidative TCA cycle, Dm I-2, chemical analysis, I2PP2A, techniques, Software, Costs and Cost Analyses, Costs, Separation, ensemble, Cost, Separations, Engineering, Cost-Minimization Analysis, Pricing, azote, sample population, Anhydrous Dextrose, l(2)k16213, nitrogeno, l(3)rK204, Computer Programs, cost, dSET/TAF-Ibeta, 2610030F17Rik, TCA cycle, Applications Softwares, Bra, assay, Cost Measure, AA407739, methodology, Glcbiochemical pathways, Metabolic Process, chemical properties, chemical characterization, degradation, Process, determination, Processes, FON1, metabolism resulting in cell growth, Metabolic Concepts, FBN, SUPERMAN, Synaptosomal-associated 25 kDa protein, Spectrum Analyses, Metabolic Processes, FLORAL ORGAN NUMBER 1, Spectrum Analysis, Cell, Biomedical, Concept, Metabolic Phenomena, ACMICD, Spectroscopy, Investigative Medicine, Metabolism Concepts, Isotope, Experimental, MS, Investigative, ECTOL1, Metabolism, chemical analysis, Medical Research, Phenomena, SUP, Mass, Concepts, GENA70, secretion, MFS1, Cell., Analysis, Metabolism Concept, Phenomenon, Medical, Metabolism Phenomena, metabolism, Investigational, Mass Spectrum Analyses, WMS, Mass Spectroscopy, WMS2, Metabolic Phenomenon, Mass Spectrum Analysis, Mass Spectrum, study, multicellular organism metabolic process, biodegradation, Analyses, Metabolic, Research, catabolism, long, Experimental Medicine, Bdr, Spectrometry, Metabolic Concept, metabolic process resulting in cell growth, FLO10, MASS, chemical content, Super protein, HERP, free, sp, SNAP-25, OCTD, Mif1, SNAP, intermediary metabolism, SSKS, Investigational Medicine, Medicine, biotransformation, assay, cellular metabolism, GPHYSD2, chemical structure, Catabolism, chemical composition, SGS, Anabolism, FLORAL DEFECTIVE 10gluco-hexose, D-Glucose, measuring, (beta-D)-Isomer, determination, Glucose, Glukose, chemical analysis., Monohydrate, (DL)-Isomer, DL-glucose, Anhydrous Dextrose, MCF7 cell, glucose, MCF7, D Glucose, scientific observation, chemical analysis, Dextrose, assay, (alpha-D)-Isomer, Glucose Monohydrate, breast carcinoma cell line, Anhydrous, MCF-7 cell, GlcfalseIsotopomer analysis of MCF-7 breast cancer cell line incubated with 13C labelled glucose (NMR assay)MS and NMR are two of the most powerful spectroscopic techniques used in the field of fluxomics, to analyze the flux distributions in cellular metabolomic pathways. Both of them possess pros and cons, such as, MS being the most sensitive and low-cost technique, while needs extensive sample processing and providing incomplete labeling data, whereas NMR needs lesser sample preparation and provides more detailed labeling data, while being less sensitive and expensive. Since both techniques could be viewed as complementary to each other, combining their data could yield a detailed and complete information about a system under study. In this case, samples were generated from a single experiment and the same set of samples was used both in MS and 1D NMR to analyze the 13C label distributions in TCA cycle intermediates. The data generated from both methods were used with a simulation script that was developed to integrate the MS and NMR data. MCF-7 breast cancer cell line was used as an experimental model and it was incubated with 100% [1,2-13C]-glucose and 100% [U-13C]-glucose for 8 hours. The cells, before and after incubation with tracers, were frozen in liquid nitrogen and then later processed for extracting the TCA cycle intermediates. Same set of samples were separated for GC-MS and NMR based analysis and processed for the respective techniques. The isotopologue analysis was performed by GC-MS and the isotopomer analysis was performed by NMR and both sets of data were integrated with the simulation software. In this study there is the data for NMR analysis. This study contains the data for NMR analysis. The GC-MS assay for this study can be found in the MetaboLights study MTBLS182. Linked Studies: <a href='https://www.ebi.ac.uk/metabolights/MTBLS182' target='_blank'>MTBLS182</a>2018-05-022015-10-01MTBLS24128272839