The targeted metabolomics analysis was performed by Metabo-Profile (Shanghai, China) as described by Xie et al.73 (link) The serum and supernatant from P. copri culture medium were extracted separately with ice-cold methanol containing internal standards. The resulting supernatants were subjected to derivatization with 3-nitrophenylhydrazine (3-NPH) and N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide (EDC)·HCl (Sigma-Aldrich, St. Louis, MO, USA). Finally, all samples were analyzed by a UPLC-MS/MS system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA), equipped with a UPLC BEH C18 column (2.1 × 100 mm, 1.7 µM). The raw data files generated by UPLC-MS/MS were processed using the Targeted Metabolome Batch Quantification (TMBQ) software (v1.0, HMI, Shenzhen, Guangdong, China) to perform peak integration, calibration, and quantitation for each metabolite. The self-developed platform iMAP (v1.0, Metabo-Profile, Shanghai, China) was used for statistical analyses, including univariate analysis (Student’s t-test or Mann – Whitney U test, as shown by volcano plot), multi-dimensional analysis (OPLS-DA), and pathway analysis, et al. Metabolites with univariate statistics p < .05 and OPLS-DA value > 1 were considered as potential markers that are responsible for the difference between groups.
N 3 dimethylamino propyl n ethylcarbodiimide edc hcl
Manufactured by Merck Group
Sourced in United States
N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide (EDC)·HCl is a water-soluble carbodiimide compound used as a crosslinking agent in chemical reactions. It is commonly employed in biomolecular conjugation and bioconjugation processes.
Automatically generated - may contain errors
Lab products found in correlation
Anhydrous pyridine, by Merck Group (2 mentions)
Mili q, by Merck Group (2 mentions)
Mili q water, by Merck Group (2 mentions)
Caproic acid, by Merck Group (2 mentions)
Isocaproic acid, by Merck Group (2 mentions)
Propionic acid, by Merck Group (2 mentions)
Acetic acid, by Merck Group (2 mentions)
Acquity uplc xevo tq s, by Waters Corporation (1 mentions)
Beh c18 column, by Waters Corporation (1 mentions)
Optima lc ms grade, by Thermo Fisher Scientific (1 mentions)
Lc ms pak filter, by Merck Group (1 mentions)
3 nitrophenylhydrazine 3 nph, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Hexamethonium, by Merck Group (1 mentions)
Urethane, by Merck Group (1 mentions)
3 nitrophenylhydrazine 3nph hcl, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Isobutyric acid, by Merck Group (1 mentions)
Valeric acid, by Merck Group (1 mentions)
5 protocols using n 3 dimethylamino propyl n ethylcarbodiimide edc hcl
1
Targeted Metabolomics Analysis of Prevelent Gut Microbiome
2
Analytical Standards Preparation and Characterization
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Formic acid was of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA). Methanol (Optima LC-MS grade), acetonitrile (Optima LC-MS grade), and isopropanol (Optima LC-MS grade) were purchased from Thermo Fisher Scientific (FairLawn, NJ, USA). Sable isotope-labeled internal standards and the derivatization regents, 3-nitrophenylhydrazine (3-NPH) and N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide (EDC)·HCl were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultra-pure water was produced by a Milli-Q system equipped with a LC-MS Pak filter (Millipore, Billerica, MA, USA).
All of the standards were purchased from TRC Chemicals (Toronto, ON, Canada), Sigma-Aldrich (St. Louis, MO, USA) and Steraloids Inc. (Newport, RI, USA). They were accurately weighed and dissolved in appropriate solutions to obtain individual stock solutions at the concentration of 5.0 mg mL− 1. Appropriate amount of each stock solution was mixed to get stock calibration solutions.
All of the standards were purchased from TRC Chemicals (Toronto, ON, Canada), Sigma-Aldrich (St. Louis, MO, USA) and Steraloids Inc. (Newport, RI, USA). They were accurately weighed and dissolved in appropriate solutions to obtain individual stock solutions at the concentration of 5.0 mg mL− 1. Appropriate amount of each stock solution was mixed to get stock calibration solutions.
3
Molecular Component Acquisition for Research
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Pyridine anhydrous, 3-nitrophenylhydrazine (3NPH·HCl), and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC·HCl) were acquired from Sigma-Aldrich (St. Louis, MO, USA). LC-MS grade acetonitrile, HPLC grade acetonitrile, HPLC grade methanol, and formic acid were obtained from J.T. Baker. Ultra-pure water (Mili-Q water) was produced by a water purification system (Mili-Q, Millipore, Milford, MA, USA).
Urethane, DMSO, BA, MOPS-PSS, BSA, ANT-3-hydroxybutyrate, hexamethonium, and all reagents used to study isolated vessels were purchased from Sigma-Aldrich, Poznan, Poland
Urethane, DMSO, BA, MOPS-PSS, BSA, ANT-3-hydroxybutyrate, hexamethonium, and all reagents used to study isolated vessels were purchased from Sigma-Aldrich, Poznan, Poland
4
Quantification of Volatile Fatty Acids
5
Quantitative Analysis of SCFAs
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All short-chain fatty acids standards ((acetic acid-AA (C2), propionic acid- PA (C3), butyric acid-BA (C4), isobutyric acid-IBA (C4), valeric acid- VA (C5), isovaleric acid-IVA (C5), caproic acid—CA (C6), isocaproic acid ICA (C6)) and short-chain fatty acid isotope-labeled standards (acetic acid-13C2, propionic acid-D6, butyric acid-13C2, isobutyric acid-D7, valeric acid-D9), pyridine anhydrous, 2-nitro phenylhydrazine (3NPH·HCl), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC·HCl)) were acquired from Sigma-Aldrich (St. Louis, MO, USA). All SCFAs stock solutions were prepared in 50% acetonitrile and stored at −20 °C. LC-MS grade acetonitrile, HPLC-grade acetonitrile, HPLC-grade methanol, and formic acid were obtained from J.T. Baker. Ultra-pure water (Mili-Q water) was produced using a water purification system (Mili-Q, Millipore, Milford, MA, USA).
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