DOI: 10.6084/m9.figshare.30209614.v2
title: Metabolomics Data Supplementary To: The pioneering gut microbiome acquired via different delivery modes in neonates shape distinct immune and metabolic environments

description: <b>Fecal liquid chromatography tandem mass spectrometry (LC-MS/MS) based metabolomics</b>To each frozen fecal pellet, 400 ul methanol was added followed by 400 ul water and 400 ul chloroform. Samples were agitated for 30 mins at 4°C then centrifuged at 16,000<i>g </i>for 20 mins. 200 ul of the aqueous layer was collected and diluted 5X in 50% methanol combined 4:1 with LC-MS/MS buffer A below prior to injection. Aqueous metabolites were separated using a Hydrophilic Interaction Liquid Chromatography method on a LD40 XR UHPLC (Shimadzu Co.) system and detected using a 6500+ QTrap mass spectrometer (AB Sciex Pte. Ltd.). A Water XBridge Amide column (3.5 μm, 3 mm X 100 mm) and metabolites were eluted using a binary gradient from A: 5% 5mM aqueous ammonium acetate pH 7.5 95% acetonitrile to B: 95 % 5 mM aqueous ammonium acetate pH 7.5 and 5% acetonitrile over 6.75 minutes Multiple reaction monitoring (MRM) ion pairs were taken from a variety of previously published methods and fidelity assessed using in-house murine organ standards.To analyze short chain fatty acids, a subaliquot of the aqueous layer was collected for derivatization of carboxylic acids with O-benzylhydroxylamine (O-BHA) according to previously established protocols. (Zeng),( Jaochico). Reaction buffer was prepared fresh consisting of 1M pyridine and 0.5 M hydrochloric acid in water. A 35 µL aliquot of the aqueous extract was taken and to the sample was added 10 µL of 1M O-BHA in reaction buffer and 10 µL of 1M 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide in reaction buffer. Samples were shaken at room temperature for 2 hrs. Each sample was quenched with 50 µL of 0.1 % formic acid for 10 min. Derivatized carboxylic acid compounds were extracted with the addition of 400 µL ethyl acetate. Samples were centrifuged at 16k xg for 5 min at 4 <sup>o</sup>C to induce layering and the upper (organic) layer was collected. The extract was dried under vacuum and each sample was resuspended in 300 µL of water for LCMS injection. Derivatized short chain fatty acid samples were analyzed using a LD40 XR UHPLC (Shimadzu Co.) system for separation and a 6500+ QTrap mass spectrometer (AB Sciex Pte. Ltd.) for detection. Samples were separated with a Waters™ Atlantis dC18 column (100Å, 3 µm, 3 mm X 100 mm) using a 6 min gradient from 5-80 % B with buffer A consisting of 0.1 % formic acid in water and B consisting of 0.1 % formic acid in methanol. Short chain fatty acids and central metabolic carboxylic acids were detected using positive mode MRMs from previously established methods and identity was confirmed by comparison to derivatized standards.Quality control samples for assessment of instrument stability were injected every 10 injections. All signals were integrated using SciexOS 3.1 (AB Sciex Pte. Ltd.). Signals with greater than 50% missing values for a specific tissue set were discarded and remaining missing values were replaced with the lowest registered signal value. Where appropriate, signals with a QC coefficient of variance greater than 30 % were discarded. Metabolites with multiple MRMs were quantified with the higher signal to noise MRM. Filtered datasets were total sum normalized after initial filtering. The short chain fatty acid dataset and the aqueous metabolomics dataset were scaled and combined using common signal for pyruvate.<br>