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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Subject:

Subject ID:	SU001911
Subject Type:	Food item
Subject Species:	-

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
GB-7	SA170629	FL020533	Ground Beef	Treatment

Collection:

Collection ID:	CO001904
Collection Summary:	Samples were homogenized, methoximated and trimethylsilylated, and untargeted metabolomic analysis was conducted via gas chromatography/electron-ionization mass spectrometry (GC/EI-MS).
Collection Protocol Filename:	protocol_burger.docx
Sample Type:	new

Treatment:

Treatment ID:	TR001924
Treatment Summary:	A novel plant-based meat alternative (n=18) and grass-fed ground beef (n=18) were matched for serving size (113 grams) and fat content (14 grams). Untargeted metabolomics was performed via gas chromatography–mass spectrometry (GC–MS) with an electron ionization (EI) by extracting ~50 mg of sample from individually cooked patties.

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

Combined analysis:

Analysis ID	AN002976
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890B
Column	Agilent DB5-MS (15m x 0.25mm,0.25um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5977
Ion Mode	NEGATIVE
Units	Log-transformed deconvoluted spectra

Chromatography:

Chromatography ID:	CH002205
Instrument Name:	Agilent 7890B
Column Name:	Agilent DB5-MS (15m x 0.25mm,0.25um)
Column Temperature:	325
Internal Standard:	TMS-D27-C14:0
Chromatography Type:	GC

MS:

MS ID:	MS002766
Analysis ID:	AN002976
Instrument Name:	Agilent 5977
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	Raw data from Agilent's MassHunter software environment were imported into the freeware, Automatic Mass Spectral Deconvolution and Identification Software or AMDIS (version 2.73)—7-9; courtesy of NIST at http://chemdata.nist.gov/mass-spc/amdis/). Peaks were not normalized, with all samples run in a single batch sequence, for which we have found normalization of peak intensities to not be necessary. Deconvoluted spectra were annotated as metabolites using an orthogonal approach that incorporates both retention time (RT) from GC and the fragmentation pattern observed in EI-MS. Peak annotation was based primarily on our own RT-locked spectral library of metabolites (2059 spectra from 1174 unique compounds at the time of analysis; January 2020). Our library is built upon the Fiehn GC/MS Metabolomics RTL Library (a gift from Agilent, their part number G1676-90000; Kind et al. 2009 3. Additional spectra have been gleaned from running pure reagent standards in our lab, from the Golm Metabolome Library10 http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/gmd.html), and from the Wiley 10th-NIST 2014 commercial library (Agilent G1730-64000). Peak alignment and chemometrics of log-base-two-transformed areas of deconvoluted peaks were performed with our own custom macros, written in our lab in Visual Basic (version 6.0) for use in the Excel (Microsoft Office Professional Plus 2019) software environment (both from Microsoft, Redmond, WA)
Ion Mode:	NEGATIVE