Uplc ms ms

For metabolomic profiling, cells were washed with PBS and snap frozen in liquid nitrogen. Samples were processed and analyzed by Metabolon. The extracts were divided into five fractions: two for analysis by two separate reverse phases (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by hydrophilic interaction chromatography/UPLC-MS/MS with negative ion mode ESI, and one sample reserved for backup. Raw data were extracted and compounds were peak identified by Metabolon. Missing values were imputed with the observed minimum after normalization. Global metabolic profiles were determined using UPLC-MS/MS (Metabolon). Shown are cell count normalized results for NAD⁺, G6P, F1.6BP, pyruvate, UDP-galactose, Gal-1-P, galactonate. The raw data obtained from our metabolome studies are listed in Dataset S3.

At age 60–64, blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes by trained research nurses (96% fasted). Samples were stored at – 80 °C.
Using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS), levels of 1401 metabolites were detected and measured by Metabolon Inc. (Durham, NC, USA) among 1814 NSHD participants. All samples were received by Metabolon at the same time point. Metabolites were assigned to nine families (lipids, amino acids, xenobiotics, peptides, nucleotides, cofactors and vitamins, carbohydrates, energy and partially characterised molecules) and further organised into pathways based on their proposed biological function informed by the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database (Supplementary Table 1). Unknown metabolites were assigned to an “Unknown” family and pathway and denoted by a number prefixed by an “X”; these were included in all analyses.
Metabolite data underwent strict quality control (QC), as detailed in [10 ], resulting in 1019 metabolites (Supplementary Notes).

Global biochemical profiles were determined by using the serum (12 mice/group) from each mouse. Metabolomic profiling analysis was performed by Metabolon (Durham, NC, USA) using ultra-high-performance liquid chromatography–tandem mass spectroscopy (UPLC-MS/MS) platforms, as previously described (15 (link)). In brief, sample (100 µl serum) preparation was conducted by using a proprietary series of organic and aqueous extractions to remove the protein fraction while allowing maximum recovery of small molecules. The resultant extract was divided into 4 fractions: one for analysis by UPLC-MS/MS (positive mode), one for UPLC-MS/MS (negative mode), one for GC-MS, and one for backup. The sample extract was dried and then reconstituted in acidic or basic liquid chromatography–compatible solvents, each of which contained ≥8 injection standards at fixed concentrations to ensure injection and chromatographic consistency. The data were extracted, with peaks and compounds identified by using the local area network backbone and database server running Oracle 10.2.0.1 Enterprise Edition (Oracle, Redwood City, CA, USA).

Cecal and plasma samples were used for untargeted metabolomics analyses by UPLC-MS/MS (Metabolon, USA). To identify potential metabolites involved in the gut-to-brain axis and contributing to the reward system, the metabolomic data were then analyzed using R and R packages “stats” and “tidyverse”. For each compound, the raw peak areas were median normalized and the minimum value was imputed for the missing values, followed by a log-transformation. Metabolites with both a fold-change > 2 and a p-value < 0.05 after the Kruskal-Wallis test were considered significant and the results were visualized with a volcano plot. Raw data obtained from untargeted metabolomic analysis in the cecal content and plasma of mice are available in Additional file 6 and 7, respectively.

Metabolomic analysis was carried out as previously described^{2 (link)}. Hearts from the sham and MI-treated groups collected after a 21-day follow-up period were shipped to Metabolon, Inc. (Durham, NC, USA), where they were stored at −80 °C until analysis. Sample preparation, quality control, UPLC‒MS/MS and data analysis were all carried out by Metabolon, Inc. A detailed description is provided in the Supplementary Materials.

One hundred flies each of 30–35 day old w¹¹¹⁸ and vaha mutants (per replicate) were collected and frozen. The samples were analyzed in triplicate by Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) by Metabolon Inc (Morrisville, NC). Lipids were extracted from samples via a modified Bligh-Dyer extraction using methanol/water/ dichloromethane in the presence of deuterated internal standards. The extracts were concentrated under nitrogen and reconstituted in 0.25 ml of 10 mM ammonium acetate dichloromethane:methanol (50:50). The extracts were transferred to inserts and placed in vials for infusion-MS analysis, performed on a Shimazdu LC with nano PEEK tubing and the Sciex SelexIon-5500 QTRAP. The samples were analyzed via both positive and negative mode electrospray. The 5500 QTRAP scan was performed in MRM mode with a total of more than 1100 MRMs. Individual lipid species were quantified by taking the peak area ratios of target compounds and their assigned internal standards, then multiplying by the concentration of internal standard added to the sample. Lipid class concentrations were calculated from the sum of all molecular species within a class.