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MB Sample ID: SA170629
Local Sample ID: | GB-7 |
Subject ID: | SU001911 |
Subject Type: | Food item |
Subject Species: | - |
Select appropriate tab below to view additional metadata details:
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 |
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 |