Summary of Study ST002391

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001538. The data can be accessed directly via it's Project DOI: 10.21228/M8WH85 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002391
Study Title	Evaluation of Two Simultaneous Metabolomic and Proteomic Extraction Protocols Assessed by Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry
Study Summary	Untargeted multi-omics analysis of plasma is an emerging tool for the identification of novel biomarkers for evaluating disease prognosis and for a better understanding of molecular mechanisms underlying human disease. The successful application of metabolomic and proteomic approaches relies on reproducibly quantifying a wide range of metabolites and proteins. Herein, we report the results of untargeted metabolomic and proteomic analyses from blood plasma samples following analyte extraction by two frequently used solvent systems: chloro-form/methanol and methanol-only. Whole blood samples were collected from participants (n=6) at University Hospital Sharjah (UHS) hospital, then plasma was separated and extracted by two methods i. methanol precipitation and, ii. 4:3 methanol:chloroform extraction. The coverage and reproducibility of the two methods were assessed by ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). The study revealed that metabolite extraction by methanol-only showed greater reproducibility for both metabolomic and proteomic quantifications than did methanol/chloroform, while yielding similar peptide coverage. However, coverage of extracted metabolites was higher with the methanol/chloroform precipitation.
Institute	Sharjah Institute for Medical Research
Last Name	Facility
First Name	Core
Address	M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah
Email	tims-tof@sharjah.ac.ae
Phone	065057656
Submit Date	2022-12-06
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2023-01-06
Release Version	1

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Project:

Project ID:	PR001538
Project DOI:	doi: 10.21228/M8WH85
Project Title:	Evaluation of Two Simultaneous Metabolomic and Proteomic Extraction Protocols Assessed by Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry
Project Summary:	Untargeted multi-omics analysis of plasma is an emerging tool for the identification of novel biomarkers for evaluating disease prognosis and for a better understanding of molecular mechanisms underlying human disease. The successful application of metabolomic and pro-teomic approaches relies on reproducibly quantifying a wide range of metabolites and proteins. Herein, we report the results of untargeted metabolomic and proteomic analyses from blood plasma samples following analyte extraction by two frequently used solvent systems: chloro-form/methanol and methanol-only. Whole blood samples were collected from participants (n=6) at University Hospital Sharjah (UHS) hospital, then plasma was separated and extracted by two methods i. methanol precipitation and, ii. 4:3 methanol:chloroform extraction. The coverage and reproducibility of the two methods were assessed by ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). The study revealed that metabolite extraction by methanol-only showed greater reproducibility for both metabolomic and proteomic quantifications than did methanol/chloroform, while yielding similar peptide coverage. However, coverage of extracted metabolites was higher with the methanol/chloroform precipitation.
Institute:	Sharjah Institute for Medical Research
Last Name:	Facility
First Name:	Core
Address:	M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah
Email:	tims-tof@sharjah.ac.ae
Phone:	065057656

Subject:

Subject ID:	SU002480
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Method
SA238175	A02-02-4395	Methanol;Chloroform
SA238176	A03-01-4396	Methanol;Chloroform
SA238177	A04-01-4398	Methanol;Chloroform
SA238178	A02-01-4394	Methanol;Chloroform
SA238179	A03-02-4397	Methanol;Chloroform
SA238180	RA04-02-4399	Methanol;Chloroform
SA238181	A01-02-4393	Methanol;Chloroform
SA238182	RB04-02-4417	Methanol;Chloroform
SA238183	RB04-01-4416	Methanol;Chloroform
SA238184	A04-02-4399	Methanol;Chloroform
SA238185	RA04-01-4398	Methanol;Chloroform
SA238186	RA03-01-4408	Methanol;Chloroform
SA238187	A05-02-4401	Methanol;Chloroform
SA238188	A06-01-4402	Methanol;Chloroform
SA238189	A01-01-4392	Methanol;Chloroform
SA238190	A06-02-4403	Methanol;Chloroform
SA238191	A05-01-4400	Methanol;Chloroform
SA238192	RA01-01-4404	Methanol;Chloroform
SA238193	RA02-02-4407	Methanol;Chloroform
SA238194	RA02-01-4406	Methanol;Chloroform
SA238195	RA01-02-4405	Methanol;Chloroform
SA238196	RA03-02-4409	Methanol;Chloroform
SA238157	B05-02-4419	Methanol Only
SA238158	B06-01-4420	Methanol Only
SA238159	B04-02-4417	Methanol Only
SA238160	B03-02-4415	Methanol Only
SA238161	B03-01-4414	Methanol Only
SA238162	B06-02-4421	Methanol Only
SA238163	B04-01-4416	Methanol Only
SA238164	RB01-01-4422	Methanol Only
SA238165	RB03-01-4426	Methanol Only
SA238166	RB03-02-4427	Methanol Only
SA238167	RB02-02-4425	Methanol Only
SA238168	RB02-01-4424	Methanol Only
SA238169	RB01-02-4423	Methanol Only
SA238170	B02-02-4413	Methanol Only
SA238171	B05-01-4418	Methanol Only
SA238172	B02-01-4412	Methanol Only
SA238173	B01-01-4410	Methanol Only
SA238174	B01-02-4411	Methanol Only

Showing results 1 to 40 of 40

Showing results 1 to 40 of 40

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.

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