Summary of Study ST003049

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 PR001899. The data can be accessed directly via it's Project DOI: 10.21228/M88147 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	ST003049
Study Title	Plasma instead of serum avoids critical confounding of clinical metabolomics studies by platelets (Part 2/3 - Eicosadomics of isolated platelets)
Study Summary	Metabolomics is an emerging and powerful molecular profiling method supporting clinical investigations. Serum and plasma are commonly used without rational prioritization. Serum is collected after blood coagulation, a complex biochemical process involving active platelet metabolism. This may affect the metabolome and increase the variance as platelet counts and function may vary substantially in individuals. A multi-omics approach systematically investigating the suitability of serum and plasma for clinical studies demonstrated that metabolites correlated well (n=461, R2=0.991), whereas lipid mediators (n=104, R2=0.906) and proteins (n=322, R2=0.860) differed substantially between specimen. Independently, analysis of platelet releasates identified most biomolecules significantly enriched in serum when compared to plasma. A prospective, randomized, controlled parallel group metabolomics trial with acetylsalicylic acid administered for 7 days demonstrated that the apparent drug effects significantly differ depending on analyzed specimen. Only serum analyses of healthy individuals suggested a significant downregulation of TXB2 and 12-HETE, which were specifically formed during coagulation in vitro. Plasma analyses reliably identified acetylsalicylic acid effects on metabolites and lipids occurring in vivo such as a decrease in polyunsaturated fatty acids. The present data suggests that plasma should be preferred above serum for clinical metabolomics studies as the serum metabolome may be substantially confounded by platelets.
Institute	University of Vienna
Department	Department of Analytical Chemistry
Laboratory	Gerner lab
Last Name	Hagn
First Name	Gerhard
Address	Währingerstraße 38, 1090 Vienna, Austria
Email	gerhard.hagn@univie.ac.at
Phone	+43 1 4277 52375
Submit Date	2024-01-22
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-04-12
Release Version	1

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Project:

Project ID:	PR001899
Project DOI:	doi: 10.21228/M88147
Project Title:	Plasma instead of serum avoids critical confounding of clinical metabolomics studies by platelets
Project Summary:	Metabolomics is an emerging and powerful molecular profiling method supporting clinical investigations. Serum and plasma are commonly used without rational prioritization. Serum is collected after blood coagulation, a complex biochemical process involving active platelet metabolism. This may affect the metabolome and increase the variance as platelet counts and function may vary substantially in individuals. A multi-omics approach systematically investigating the suitability of serum and plasma for clinical studies demonstrated that metabolites correlated well (n=461, R2=0.991), whereas lipid mediators (n=104, R2=0.906) and proteins (n=322, R2=0.860) differed substantially between specimen. Independently, analysis of platelet releasates identified most biomolecules significantly enriched in serum when compared to plasma. A prospective, randomized, controlled parallel group metabolomics trial with acetylsalicylic acid administered for 7 days demonstrated that the apparent drug effects significantly differ depending on analyzed specimen. Only serum analyses of healthy individuals suggested a significant downregulation of TXB2 and 12-HETE, which were specifically formed during coagulation in vitro. Plasma analyses reliably identified acetylsalicylic acid effects on metabolites and lipids occurring in vivo such as a decrease in polyunsaturated fatty acids. The present data suggests that plasma should be preferred above serum for clinical metabolomics studies as the serum metabolome may be substantially confounded by platelets.
Institute:	University of Vienna
Department:	Department of Analytical Chemistry
Laboratory:	Gerner lab
Last Name:	Hagn
First Name:	Gerhard
Address:	Währingerstraße 38, 1090 Vienna, Austria
Email:	gerhard.hagn@univie.ac.at
Phone:	+43 1 4277 52375
Publications:	https://doi.org/10.1021/acs.jproteome.3c00761

Subject:

Subject ID:	SU003164
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

Factors:

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

mb_sample_id	local_sample_id	Treatment
SA331236	Platelets_Donor_10_con_1	Control
SA331237	Platelets_Donor_9_con_1	Control
SA331238	Platelets_Donor_8_con_1	Control
SA331239	Platelets_Donor_10_con_2	Control
SA331240	Platelets_Donor_8_con_2	Control
SA331241	Platelets_Donor_11_con_2	Control
SA331242	Platelets_Donor_1_con_1	Control
SA331243	Platelets_Donor_12_con_2	Control
SA331244	Platelets_Donor_12_con_1	Control
SA331245	Platelets_Donor_7_con_2	Control
SA331246	Platelets_Donor_11_con_1	Control
SA331247	Platelets_Donor_9_con_2	Control
SA331248	Platelets_Donor_3_con_1	Control
SA331249	Platelets_Donor_3_con_2	Control
SA331250	Platelets_Donor_2_con_2	Control
SA331251	Platelets_Donor_7_con_1	Control
SA331252	Platelets_Donor_1_con_2	Control
SA331253	Platelets_Donor_4_con_1	Control
SA331254	Platelets_Donor_2_con_1	Control
SA331255	Platelets_Donor_6_con_2	Control
SA331256	Platelets_Donor_4_con_2	Control
SA331257	Platelets_Donor_5_con_2	Control
SA331258	Platelets_Donor_6_con_1	Control
SA331259	Platelets_Donor_5_con_1	Control
SA331260	Platelets_Donor_9_act_1	Platelet activation
SA331261	Platelets_Donor_9_act_2	Platelet activation
SA331262	Platelets_Donor_8_act_2	Platelet activation
SA331263	Platelets_Donor_8_act_1	Platelet activation
SA331264	Platelets_Donor_10_act_1	Platelet activation
SA331265	Platelets_Donor_12_act_1	Platelet activation
SA331266	Platelets_Donor_7_act_2	Platelet activation
SA331267	Platelets_Donor_12_act_2	Platelet activation
SA331268	Platelets_Donor_11_act_2	Platelet activation
SA331269	Platelets_Donor_11_act_1	Platelet activation
SA331270	Platelets_Donor_10_act_2	Platelet activation
SA331271	Platelets_Donor_1_act_1	Platelet activation
SA331272	Platelets_Donor_3_act_1	Platelet activation
SA331273	Platelets_Donor_3_act_2	Platelet activation
SA331274	Platelets_Donor_2_act_2	Platelet activation
SA331275	Platelets_Donor_2_act_1	Platelet activation
SA331276	Platelets_Donor_1_act_2	Platelet activation
SA331277	Platelets_Donor_4_act_1	Platelet activation
SA331278	Platelets_Donor_4_act_2	Platelet activation
SA331279	Platelets_Donor_6_act_2	Platelet activation
SA331280	Platelets_Donor_6_act_1	Platelet activation
SA331281	Platelets_Donor_5_act_2	Platelet activation
SA331282	Platelets_Donor_5_act_1	Platelet activation
SA331283	Platelets_Donor_7_act_1	Platelet activation

Showing results 1 to 48 of 48

Showing results 1 to 48 of 48

Collection:

Collection ID:	CO003157
Collection Summary:	PLATELETS: Whole blood of six healthy donors (three male and three female) in the age range of 26 to 51 years were collected in biological duplicates with one week in between the donations. Each donor gave written consent and the study was approved by the ethics committee of the Medical University of Vienna in accordance with the Declaration of Helsinki (EC 1430/2018). No medical substances interfering with the normal physiology of platelets such as aspirin, paracetamol or ibuprofen were taken by the donors 48 hours prior to blood donation. Two CPDA (citrate-phosphate-dextrose-adenine)-S-Monovette tubes (Sarstedt) of venous blood were collected per donor and donation. To isolate platelet rich plasma (PRP), the tubes were centrifuged for 20 min at 100 g with acceleration and deceleration set to 4. To purify platelets, size exclusion chromatography using 2 % B agarose beads (50-150 μm; abtbeads.es) was performed. Therefore, columns were equipped with a cotton frit and 20 mL of reconstituted agarose bead solution diluted 1:2 in RPMI medium (1X with L-Glutamine; Gibco, Thermo Fischer Scientific, Austria). Columns were washed with 2 mL RPMI medium before 1 ml of PRP was carefully pipetted to the column and topped with RPMI. The fraction containing purified platelets was collected and divided in two aliquots, one for platelet activation and one serving as control. To achieve platelet activation, ionomycin calcium salt (Sigma-Aldrich) was added to one aliquot to a final concentration of 1 μM. All samples were incubated for 15 min at room temperature before centrifugation at 2000 g for 5 min.
Sample Type:	Blood (isolated platelets)

Treatment:

Treatment ID:	TR003173
Treatment Summary:	To achieve platelet activation, ionomycin calcium salt (Sigma-Aldrich) was added to one aliquot to a final concentration of 1 μM. All samples were incubated for 15 min at room temperature before centrifugation at 2000 g for 5 min.

Sample Preparation:

Sampleprep ID:	SP003170
Sampleprep Summary:	PLATELETS: The supernatant was transferred into new tubes and protein precipitation was performed by adding ice cold ethanol (LC-MS grade) in a ratio of 1:5. Additionally, 5 μL of an internal standard mixture of 12S‐HETE‐d8, 15S‐HETE‐d8, 5‐Oxo‐eicosatetraenoic acid (ETE)‐d7, 11,12‐dihydroxy-5Z,8Z,14Z-eicosatrienoic acid (DiHETrE)‐d11, PGE2‐d4 and 20‐HETE‐d6 (concentration per sample can be found above) were added to each sample. Samples were then stored at -20°C. After overnight precipitation, samples were centrifuged for 30 min at 4536 g at +4°C. The supernatant was then transferred into new 15 mL FalconTM tubes. EtOH was evaporated via vacuum centrifugation at 37°C until the original sample volume was restored. For solid phase extraction (SPE) samples were loaded onto preconditioned StrataX SPE columns (30 mg mL-1; Phenomenex, Torrance, CA, USA) using Pasteur pipettes. After sample loading, the SPE columns were washed with 5 mL of MS grade water and eluted with ice-cold MeOH (500 µL; MeOH abs.; VWR International, Vienna, Austria) containing 2% formic acid (FA; Sigma-Aldrich). MeOH was evaporated using a gentle nitrogen stream at room temperature and the dried samples were reconstituted in 150 µL reconstitution buffer (H2O:ACN:MeOH + 0.2% FA–vol% 65:31.5:3.5). The samples were then transferred into an autosampler held at stored at 4°C and subsequently measured via LC-MS/MS. 12S-HETE-d8: 6.67 pg/µL 15S-HETE-d8: 6.67 pg/µL 5-Oxo-ETE-d7: 20 pg/µL 11,12-DiHETrE-d11: 6.67 pg/µL PGE2-d4: 13.33 pg/µL 20-HETE-d6: 6.67 pg/µL

Sampleprep ID:

SP003170

Sampleprep Summary:

PLATELETS: The supernatant was transferred into new tubes and protein precipitation was performed by adding ice cold ethanol (LC-MS grade) in a ratio of 1:5. Additionally, 5 μL of an internal standard mixture of 12S‐HETE‐d8, 15S‐HETE‐d8, 5‐Oxo‐eicosatetraenoic acid (ETE)‐d7, 11,12‐dihydroxy-5Z,8Z,14Z-eicosatrienoic acid (DiHETrE)‐d11, PGE2‐d4 and 20‐HETE‐d6 (concentration per sample can be found above) were added to each sample. Samples were then stored at -20°C. After overnight precipitation, samples were centrifuged for 30 min at 4536 g at +4°C. The supernatant was then transferred into new 15 mL FalconTM tubes. EtOH was evaporated via vacuum centrifugation at 37°C until the original sample volume was restored. For solid phase extraction (SPE) samples were loaded onto preconditioned StrataX SPE columns (30 mg mL-1; Phenomenex, Torrance, CA, USA) using Pasteur pipettes. After sample loading, the SPE columns were washed with 5 mL of MS grade water and eluted with ice-cold MeOH (500 µL; MeOH abs.; VWR International, Vienna, Austria) containing 2% formic acid (FA; Sigma-Aldrich). MeOH was evaporated using a gentle nitrogen stream at room temperature and the dried samples were reconstituted in 150 µL reconstitution buffer (H2O:ACN:MeOH + 0.2% FA–vol% 65:31.5:3.5). The samples were then transferred into an autosampler held at stored at 4°C and subsequently measured via LC-MS/MS. 12S-HETE-d8: 6.67 pg/µL 15S-HETE-d8: 6.67 pg/µL 5-Oxo-ETE-d7: 20 pg/µL 11,12-DiHETrE-d11: 6.67 pg/µL PGE2-d4: 13.33 pg/µL 20-HETE-d6: 6.67 pg/µL

Combined analysis:

Analysis ID	AN005000
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Phenomenex Kinetex XB-C18(150 * 2.1 mm, 2.6um, 100 A°)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	NEGATIVE
Units	normalized AUC

Chromatography:

Chromatography ID:	CH003778
Chromatography Summary:	For LC-MS analyses, analytes were separated using a Thermo Scientific Vanquish (UHPLC) system equipped with a Kinetex C18-column (2.6 µm, XB-C18, 100 A° , LC Column 150 * 2.1 mm; Phenomenex) applying a gradient flow profile (mobile phase A: H2O + 0.2% FA, mobile phase B: ACN:MeOH (vol% 90:10) + 0.2% FA) starting at 35% B and increasing to 90% B (1–10 min), further increasing to 99% B within 0.5 min and held for 5 min. Solvent B was then decreased to the initial level of 35% within 0.5 min and the column was equilibrated for 4 min, resulting in a total run time of 20 min. The flow rate was kept at 200 µL min-1 and the column oven temperature at 40°C. The injection volume was 20 µL and all samples were analysed in technical duplicates.
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex XB-C18(150 * 2.1 mm, 2.6um, 100 A°)
Column Temperature:	40
Flow Gradient:	0min with 35% B to 90% B (1–10 min), further increasing to 99% B within 0.5 min and held for 5 min. Solvent B was then decreased to the initial level of 35% within 0.5 min and the column was equilibrated for 4 min, resulting in a total run time of 20 min.
Flow Rate:	0.2 mL/min
Solvent A:	100% water; 0.2% formic acid
Solvent B:	90% acetonitrile/10% methanol; 0.2% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004740
Analysis ID:	AN005000
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The Vanquish UHPLC system was coupled to a Q ExactiveTM HF Quadrupole-OrbitrapTM high-resolution mass spectrometer (Thermo Fisher Scientific, Austria), equipped with a HESI source for negative ionization to perform the mass spectrometric analysis. The MS scan range was 250-700 m/z with a resolution of 60,000 (at m/z 200) on the MS1 level. A Top 2 method was applied for fragmentation (HCD 24 normalized collision energy), preferable 33 m/z values specific for well-known eicosanoids and precursor molecules from an inclusion list. The resulting fragments were analysed on the MS2 level at a resolution of 15,000 (at m/z 200). Operating in negative ionization mode, a spray voltage of 3.5 kV and a capillary temperature of 253°C were applied. Sheath gas was set to 46 and the auxiliary gas to 10 (arbitrary units).
Ion Mode:	NEGATIVE