DOI: 10.25345/c5gq6r592
title: MassIVE MSV000089224 - Analysis of dietary 13C6 isotope tracing through untargeted metabolomics - Reversephasepositive part
title: Methods 13C6 Lysine isotope labeling study. Male Bl6/N mice (12 weeks old) were fed a custom diet (Silantes) containing more than 99% 13C6 lysine. The protocol for labeling was described previously. After 1, 2, and 8 weeks (protocol 1) or 1, 2, and 3 weeks (protocol 2), mice were sacrificed and perfused with ice-cold PBS. All denominated organs were snap-frozen from the same mouse and stored at -80C degrees. Blood was separated in erythrocyte and plasma by centrifugation. Metabolomics sample preparation. Frozen organs were kept on dry ice and 10 mg tissue was weighed. Then, 800 ul ice-cold extraction solution containing Acetonitril : Methanol : Water 2:2:1 was added. Samples were subjected to tissue homogenization using a multiplex-bead-beater (Storm) for 30 sec (liver), 1 min (lung), or 45 sec (all other tissues). The supernatant was transferred to a prechilled Eppendorf tube. Then, beads were washed with 200 ul of the extraction solution, and the wash solution was added to the remaining homogenate. The homogenate was incubated at -20C degrees for 2 hrs. Then, tissue was spun down at 4 C degrees for 20 min, and the supernatant was transferred to another vial. The supernatant containing the extract was transferred into a speed vac and dried down. The next morning, the dried-down extract was once resuspended in 100 ul (per 10 mg tissue) of acetonitrile-water 1:1, and the solution was centrifuged at 4 C degrees. Then, the solution was transferred to autosampler vials and stored at -80 C until further use. Untargeted metabolomics analysis and mass spectrometry. LC-MS/MS analysis for metabolomics was performed as previously described. Data was annotated with in-source fragments and adducts. The mass spectrometer was initially calibrated using NaFormate peaks and in addition post-run. For untargeted metabolomic analysis, we used a UHPLC-MS approach. For fractionation, we used hydrophilic interaction liquid chromatography (HILIC) fractionation and reversed-phase (RP) chromatography as previously described22. We used a quadrupole time-of-flight instrument (Impact II, Bruker, Bremen, Germany) coupled to a ultrahigh-performance liquid chromatography (UHPLC) device (Bruker Elute, Bruker, Billerica, MA), or to an Agilent Infinity 1290 UHPLC device (Agilent, USA). The MS was calibrated using sodium formate (post-run mass calibration). Data were acquired over an m/z range of 50 to 1000 Da in positive ion mode and negative ion mode (HILIC only). Electrospray source conditions were set as follows: end plate offset, 500 V; dry gas temperature, 200C; drying gas, 6 liters/min; nebulizer, 1.6 bar; and capillary voltage, 3500 V. To increase metabolome coverage and minimize ion suppression, we used a dual fractionation strategy. For RP separation, an ACQUITY BEH C18 column (1.0 x 100 mm, 1.7-um particle size; Waters Corporation, Milford, MA) was used, and for HILIC fractionation, a ACQUITY BEH amide (1.0 x 100 mm, 1.7-um particle size; Waters Corporation, Milford, MA) column was used. Flow was 150 ul/min, and a binary buffer system consisting of buffer A (0.1% FA) and buffer B (0.1% FA in acetonitrile) was used. The gradient for RP was: 99% A for 1 min, 1% A over 9 min, 35% A over 13 min, 60% A over 3 min, and held at 60% A for an additional 1 min. The gradient for HILIC consisted of 1% A for 1 min, 35% A over 13 min, 60% A over 3 min, and held at 60% A for an additional 1 min. The injection volume was always 2 ul. For molecule identification purposes, putative molecules of interest were fragmented using three different collision energies (10, 20 eV) or ramp collision energies (20 to 50 eV). Untargeted metabolomics data analysis. Bruker Raw files (*.d) were transformed into mzml files using the compassxport_converter.py script (Bruker). Then, data files were uploaded to XCMS online, and differential peaks were extracted in positive and negative ion mode50. Feature Detection was performed with the centwave method with the following options: ppm =10, minimum peak width = 5, maximum peak width = 20, mzdiff = 0.01, Signal/Noise = 6, Integration method =1, prefilter peaks =3, prefilter intensity = 100, noise filter = 100. For Retention time correction, we used obiwarp method with profStep = 1. Alignment was perfomed with bw 05, minfrac = 0.5, mzwid = 0.015. mminsamp =1, and max = 100. Statistical test was performed using an unpaired parametric t-test (Welsh), with post-hoc analysis = TRUE,. Statistical filtering was performed for priorization of features of interest, including a fold change of at least 1.5, and a p-value lower than 0.05, and a corrected p value (q-value) lower than 0.05. The analysis included PCA and multivariate analyses. Metabolites were identified based on 1) unique mass, 2) MS2 spectra comparison to authentic standard 3) coelution with authentic standard, and 4) isotopic pattern. The following adducts were routinely considered : Na, H, for positive mode, and Cl, formate for negative mode.