Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA170629

Local Sample ID:	GB-7
Subject ID:	SU001911
Subject Type:	Food item
Subject Species:	-

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Sample Preparation:

Sampleprep ID:	SP001917
Sampleprep Summary:	One-gram microcore samples were obtained from the middle of each patty using a bioptome device, immediately frozen in liquid nitrogen, and stored at -80 degrees °C until metabolomics analysis. Microcore samples the plant-based meat replacement and bovine skeletal muscle (i.e., beef) were powdered under liquid N2 and homogenized in 50% aqueous acetonitrile containing 0.3% formic acid (50 mg wet weight sample per ml homogenate) using a Qiagen Retsch Tissue Lyser II set to a frequency of 30 oscillations/sec for a total of 2 min with one 5 mm glass ball (GlenMills, Inc, #7200-005000TM) per tube. Proteins in sample homogenates were subsequently “crash precipitated with 750 µl dry methanol and centrifuged at 13.500 x g rcf for 5 minutes (Vial CentrifugeTM, MicroSolv, catalog C2417) and spiked with D27-deuterated myristic acid (D27-C14:0) (Sigma 366889, 6.25 mg/liter) for retention-time locking (described below). Methanolic extracts were dried in a Savant SPD111V SpeedVac Concentrator (Thermo Scientific, Asheville, NC). 25 µl methoxyamine hydrochloride (18 mg/ml in dry pyridine: Fisher Scientific, catalog number T324-50) was then added to each sample and incubated at 50 °C for 30 minutes for methoximation of certain reactive carbonyl groups. Finally, metabolites were rendered volatile by replacement of easily exchangeable protons with trimethylsilyl (TMS) groups using N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA; 75 µl per sample Cerilliant M-132, Sigma, St. Louis, MO) at 50 ºC for 30 minutes. Biological comparators (beef vs. plant-meat based alternative) were run in direct succession (e.g., the order A-B-A-B) on a 7890B GC / 5977B single-quadrupole, Inert MS (Agilent Technologies, Santa Clara, CA). Prior to each daily run (2 total), the starting inlet pressure was empirically adjusted such that the retention time of the TMS-D27-C14:0 standard is set at ~16.727 minutes. Radical cations generated with conventional electron ionization via a tungsten-rhenium filament set to an energy of 70 eV were scanned broadly from 600 to 50 m/z in the detector throughout the run

Sampleprep ID:

SP001917

Sampleprep Summary:

One-gram microcore samples were obtained from the middle of each patty using a bioptome device, immediately frozen in liquid nitrogen, and stored at -80 degrees °C until metabolomics analysis. Microcore samples the plant-based meat replacement and bovine skeletal muscle (i.e., beef) were powdered under liquid N2 and homogenized in 50% aqueous acetonitrile containing 0.3% formic acid (50 mg wet weight sample per ml homogenate) using a Qiagen Retsch Tissue Lyser II set to a frequency of 30 oscillations/sec for a total of 2 min with one 5 mm glass ball (GlenMills, Inc, #7200-005000TM) per tube. Proteins in sample homogenates were subsequently “crash precipitated with 750 µl dry methanol and centrifuged at 13.500 x g rcf for 5 minutes (Vial CentrifugeTM, MicroSolv, catalog C2417) and spiked with D27-deuterated myristic acid (D27-C14:0) (Sigma 366889, 6.25 mg/liter) for retention-time locking (described below). Methanolic extracts were dried in a Savant SPD111V SpeedVac Concentrator (Thermo Scientific, Asheville, NC). 25 µl methoxyamine hydrochloride (18 mg/ml in dry pyridine: Fisher Scientific, catalog number T324-50) was then added to each sample and incubated at 50 °C for 30 minutes for methoximation of certain reactive carbonyl groups. Finally, metabolites were rendered volatile by replacement of easily exchangeable protons with trimethylsilyl (TMS) groups using N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA; 75 µl per sample Cerilliant M-132, Sigma, St. Louis, MO) at 50 ºC for 30 minutes. Biological comparators (beef vs. plant-meat based alternative) were run in direct succession (e.g., the order A-B-A-B) on a 7890B GC / 5977B single-quadrupole, Inert MS (Agilent Technologies, Santa Clara, CA). Prior to each daily run (2 total), the starting inlet pressure was empirically adjusted such that the retention time of the TMS-D27-C14:0 standard is set at ~16.727 minutes. Radical cations generated with conventional electron ionization via a tungsten-rhenium filament set to an energy of 70 eV were scanned broadly from 600 to 50 m/z in the detector throughout the run