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MB Sample ID: SA238183
Local Sample ID: | RB04-01-4416 |
Subject ID: | SU002480 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002480 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
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RB04-01-4416 | SA238183 | FL029594 | Methanol;Chloroform | Method |
Collection:
Collection ID: | CO002473 |
Collection Summary: | Human whole blood samples (5mL) were collected from healthy males and females (n=6) via venipuncture into EDTA tube at University Hospital Sharjah. All participants gave in-formed consent for inclusion before enrollment in the study. Blood was centrifuged (14000 rpm for 15 minutes at 24°C) to separate plasma samples, which were stored at -80°C. |
Sample Type: | Blood (plasma) |
Treatment:
Treatment ID: | TR002492 |
Treatment Summary: | No treatment was applied, we were comparing extraction methods. |
Sample Preparation:
Sampleprep ID: | SP002486 |
Sampleprep Summary: | Sample preparation 2.3.1Methanol precipitation for metabolomic extraction. 100 µL from each plasma sample was transferred to Eppendorf, then a volume of 300 µL of methanol was added to the aliquot. Samples were vortexed, then chilled at -20 °C for 2 hours followed by centrifugation (14000 rpm, 15 minutes, 24°C) to precipitate protein. The metabolite-containing supernatants were collected and transferred to glass vials for dry-ing using the EZ-2 Plus (GeneVac, Ipswich, UK) at 40 ±1°C and the protein pellets that remained were air dried for proteomics (see section 2.4). Dried metabolite samples were resuspended with 200 µl (0.1% formic acid in water), and vortexed for 2 min. The samples were filtered using a hydrophilic nylon syringe filter (0.45 μm pore size) and placed with-in glass inserts prior to being analyzed by Q-TOF MS. 4.3.2 Methanol: chloroform for metabolomic extraction. 100 µL from each sample was transferred to Eppendorf, then a volume of 400 µL of methanol and 300 µL of chloroform was added to the aliquot. Samples were vortexed then centrifuged (14000 rpm, 15 minutes, 24°C). Two metabolite-containing layers, an upper aqueous- and lower organic phase, were obtained separated by a thin white proteinaceous disc. The upper layer for each sample was transferred to glass vials and a volume of 400 µL of methanol was added, followed by vortexing and centrifugation to pellet the protein disc, after which the remaining supernatant was transferred to the same glass vials as be-fore for drying step and the protein pellets that remained were air dried for proteomics. Dried metabolomics samples were resuspended with 200 µL (0.1% formic acid in water) to be injected into HPLC and analyzed by Q-TOF MS. |
Combined analysis:
Analysis ID | AN003896 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Bruker Elute |
Column | Hamilton Intensity Solo 2 C18(100 x 2.1 mm,1.8um) |
MS Type | ESI |
MS instrument type | QTOF |
MS instrument name | Bruker timsTOF |
Ion Mode | POSITIVE |
Units | AU |
Chromatography:
Chromatography ID: | CH002885 |
Instrument Name: | Bruker Elute |
Column Name: | Hamilton Intensity Solo 2 C18(100 x 2.1 mm,1.8um) |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS003636 |
Analysis ID: | AN003896 |
Instrument Name: | Bruker timsTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | Metabolomics Data Processing. For metabolomic analysis, MetaboScape® 4.0 program (Bruker Daltonics, Billerica, MA, USA) was employed for data processing, feature extrac-tion and metabolite identification. The T-ReX 2D/3D workflow was used to identify the molecular features with the following settings: The minimum peak length was set to 7 spectra and the minimum intensity threshold was 1000 counts for peaks detection. The peak area was employed for quantification and the injected external calibrant in the in-terval of 0–0.3 min was used to recalibrate the mass spectra. The selected mass to charge ratio (m/z) and retention time for scanning were in the ranges of 20–1300 m/z and 0.3–30 min, respectively. MS/MS spectra for features were averaged on import and features found at least in 12 of the 40 injections were taken into further consideration. Metabolites were identified by matching to the human metabolome database (HMDB) by combined MS/MS, precursor m/z values, and isotopic pattern scores. Where multiple features matched a given database entry, the annotation quality score (AQ score) was used to select only the best matching feature. |
Ion Mode: | POSITIVE |