Supporting data for bariatric surgery patient fecal metabolomics and bile acid measurements

Source data is included for targeted metabolomics analysis of fecal samples from bariatric surgery patients pre and post intervention. The attached data consists of two sets. The first is a total sum normalized polar metabolomics dataset from three separate liquid chromatography tandem mass spectrometry (LC-MS/MS) methods that have been scaled together via common signals between methods. The second dataset is a targeted bile acid data also collected via LC-MS/MS methods that has been normalized to the total signal abundance from the polar metabolomics dataset. Details on sample preparation and data acquisition are included below. <b>Metabolite and Lipid Sample Preparation</b>For all LCMS methods, LCMS-grade solvents were used. For bile acid analysis 400 µL of homogenized feces was taken from the fecal collection tubes and added to 500 µL of ice-cold methanol. To each sample 5 µL of the Bile Acid SPLASH® (Avanti Polar Lipids Inc.) and 2 µg of butylated hydroxytoluene was added. Samples were agitated via shaking at 4 ^oC for 20 minutes and then centrifuged at 16k xg for 20 min. An aliquot of the supernatant was taken directly for liquid chromatograph mass spectrometry (LCMS) analysis.For short chain fatty acid (SCFA) and polar metabolomics a separate 400 µL aliquot of homogenized feces was added to 400 µL of water. Following mixing, 400 µL of chloroform was added. Samples were shaken for 30 minutes at 4 ^oC and subsequently centrifuged at 16k xg for 20 min. 400 µL of the top (aqueous) layer was collected. The aqueous layer was sub-aliquoted for SCFA derivatization or diluted 5x in 50% methanol in water and prepared for LCMS injection.<b>Short Chain Fatty Acid Derivatization</b>To preserve SCFA’s for analysis an aliquot of the aqueous fraction was derivatized with O-benzylhydroxylamine (O-BHA) as previously described with modifications (https://doi.org/10.1016/j.jchromb.2018.02.040, https://doi.org/10.4155/bio-2018-0241). Reaction buffer contained 1M pyridine and 0.5 M hydrochloric acid in water. To 35 µL of sample, 10 µL of 1M O-BHA in reaction buffer and 10 µL of 1M 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide in reaction buffer were added. Samples were derivatized for 2 hours at room temperature with constant agitation. Addition of 50 µL of 0.1 % formic acid was used to quench the reaction. To extract derivatized molecules 400 µL of ethyl acetate was added. The samples were centrifuged at 16k xg and 4 ^oC for 5 min to induce layering. The upper (organic) layer was collected and dried under vacuum. Samples were resuspended in 300 µL of water for LCMS injection.<b>Liquid Chromatography Mass Spectrometry</b>Tributylamine and all synthetic molecular references were purchased from Millipore Sigma. LCMS grade water, methanol, isopropanol and acetic acid were purchased through Fisher Scientific.All samples were separated using a Sciex ExionLC™ AC system and measured using a Sciex 5500 QTRAP® or Sciex 6500+ QTRAP® mass spectrometer.Polar metabolites were analyzed as previously described (https://doi.org/10.1007/s11306-015-0790-y, https://doi.org/10.1016/j.jbc.2023.105319). For all metabolomics analysis, quality control samples, consisting of a mixture of the analyzed samples, were injected after every 10 injections to control for signal stability. Sample were analyzed via separate negative ionization and positive ionization methods. For negative mode analysis, a Waters Atlantis T3 column (100 Å, 3 μm, 3 mm × 100 mm) with a gradient from 5 mM tributylamine, 5 mM acetic acid in 2% isopropanol, 5% methanol, 93% water (v/v) to 100% isopropanol over 15 min was used. For positive mode analysis, samples were separated on a Phenomenex Kinetex F5 column (100 Å, 2.6 μm, 2.1 mm × 100 mm) column with a gradient from 100% water with 0.1% formic acid to 95% acetonitrile with 0.1% formic acid over 5 min. Each metabolite was measured using at two distinct multiple reaction monitoring (MRM) signals and a defined retention time.For SCFA analysis samples were separated on a Waters™ Atlantis dC18 column (100Å, 3 µm, 3 mm X 100 mm) with a 6 min gradient from 5-80 % B with buffer A as 0.1 % formic acid in water and B as 0.1 % formic acid in methanol. All SCFA were measured using positive ionization using MRMs that featured the characteristic 91 daughter ion from O-BHA derivatization. Identity was confirmed via comparison to previous standards.Bile acid samples were separated on a Kinetex® Polar C18 (100Å, 2.6 µm, 3 mm X 100 mm) using a binary gradient of A: 0.01 % acetic acid in water and B: 0.01 % acetic acid in methanol. A 20 min gradient from 40-100 % B was utilized for separation. Samples were detected in negative MRM mode using previous validated MRMs (https://doi.org/10.3390/metabo10010026). Internal bile acid standard signals were used to confirm signal identities and retention times.All signals were integrated using SciexOS 3.1 (AB Sciex Pte. Ltd.). Signals with greater than 50% missing values were discarded and remaining missing values were replaced with the lowest registered signal value. All 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 prior to analysis. The SCFA dataset and the two polar metabolomics datasets were scaled and combined using common signal for serine and succinate for the positive mode metabolite method and the SCFA method respectively. Bile acid data was normalized to the total polar metabolite signal in each sample and all analysis was conducted via conversion ratios between bile acids signals within a patient. A paired t-test was used for all bile acid and metabolite statistics and a Benjamini-Hochberg method for correction for multiple comparisons was imposed where indicated.<br>