Summary of study ST001710

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 PR001095. The data can be accessed directly via it's Project DOI: 10.21228/M85976 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.

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Study IDST001710
Study TitleMetabolic signatures of NAFLD - Lipidomics data (part 1 of 3)
Study SummarySerum samples were randomized and extracted using a modified version of the previously-published Folch procedure, as applied recently [20]. The maternal samples were analysed as one batch and the cord blood samples as a second batch. In short, 10 µL of 0.9% NaCl and, 120 µL of CHCl3: MeOH (2:1, v/v) containing the internal standards (c = 2.5 µg/mL) was added to 10 µL of each serum sample. The standard solution contained the following compounds: 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (PE(17:0/17:0)), N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (SM(d18:1/17:0)), N-heptadecanoyl-D-erythro-sphingosine (Cer(d18:1/17:0)), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC(17:0/17:0)), 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(17:0)) and 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (PC(16:0/d31/18:1)), were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and, triheptadecanoylglycerol (TG(17:0/17:0/17:0)) was purchased from Larodan AB (Solna, Sweden). The samples were vortex mixed and incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min). 60 µL from the lower layer of each sample was then transferred to a glass vial with an insert and 60 µL of CHCl3: MeOH (2:1, v/v) was added to each sample. The samples were stored at -80 °C until analysis. Calibration curves using 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(16:0e/18:1(9Z))), 1-(1Z-octadecenyl)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(18:0p/18:1(9Z))), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:0)), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:1)), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)), 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PC(18:0p/22:6)) and 1-stearoyl-2-linoleoyl-sn-glycerol (DG(18:0/18:2)), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (LPE(18:1)), N-(9Z-octadecenoyl)-sphinganine (Cer(d18:0/18:1(9Z))), 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)) from Avanti Polar Lipids, 1-Palmitoyl-2-Hydroxy-sn-Glycero-3-Phosphatidylcholine (LPC(16:0)), 1,2,3 trihexadecanoalglycerol (TG(16:0/16:0/16:0)), 1,2,3-trioctadecanoylglycerol (TG(18:0/18:0/18:)) and 3β-hydroxy-5-cholestene-3-stearate (ChoE(18:0)), 3β-Hydroxy-5-cholestene-3-linoleate (ChoE(18:2)) from Larodan, were prepared to the following concentration levels: 100, 500, 1000, 1500, 2000 and 2500 ng/mL (in CHCl3:MeOH, 2:1, v/v) including 1250 ng/mL of each internal standard. The samples were analyzed by ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS). Briefly, the UHPLC system used in this work was a 1290 Infinity II system from Agilent Technologies (Santa Clara, CA, USA). The system was equipped with a multi sampler (maintained at 10 °C), a quaternary solvent manager and a column thermostat (maintained at 50 °C). Injection volume was 1 µL and the separations were performed on an ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm) by Waters (Milford, MA, USA). The mass spectrometer coupled to the UHPLC was a 6545 QTOF from Agilent Technologies interfaced with a dual jet stream electrospray (Ddual ESI) ion source. All analyses were performed in positive ion mode and MassHunter B.06.01 (Agilent Technologies) was used for all data acquisition. Quality control was performed throughout the dataset by including blanks, pure standard samples, extracted standard samples and control serum samples, including in-house serum and a pooled QC with an aliquot of each sample was collected and pooled and used as quality control sample. Relative standard deviations (% RSDs) for identified lipids in the control serum samples (n = 13) and in the pooled serum samples (n = 54) were on average 22.4% and 17.5%, respectively.
Institute
Örebro University
Last NameMcGlinchey
First NameAidan
AddressSchool of Medical Sciences, Örebro, Örebro, 70281, Sweden
Emailaidan.mcglinchey@oru.se
Phone+46736485638
Submit Date2021-02-10
Raw Data AvailableYes
Raw Data File Type(s).mzML
Analysis Type DetailLC-MS
Release Date2021-05-01
Release Version1
Aidan McGlinchey Aidan McGlinchey
https://dx.doi.org/10.21228/M85976
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR001095
Project DOI:doi: 10.21228/M85976
Project Title:Metabolomic signatures of NAFLD
Project Summary:Background and Aims: Nonalcoholic fatty liver disease (NAFLD) is a progressive liver disease that is strongly associated with type 2 diabetes. Accurate, non-invasive diagnostic tests to delineate the different stages: degree of steatosis, grade of nonalcoholic steatohepatitis (NASH) and stage fibrosis represent an unmet medical need. In our previous studies, we successfully identified specific serum molecular lipid signatures which associate with the amount of liver fat as well as with NASH. Here we report underlying associations between clinical data, lipidomic profiles, metabolic profiles and clinical outcomes, including downstream identification of potential biomarkers for various stages of the disease. Method: We leverage several statistical and machine-learning approaches to analyse clinical, lipidomic and metabolomic profiles of individuals from the European Horizon 2020 project: Elucidating Pathways of Steatohepatitis (EPoS). We interrogate data on patients representing the full spectrum of NAFLD/NASH derived from the EPoS European NAFLD Registry (n = 627). We condense the EPoS lipidomic data into lipid clusters and subsequently apply non-rejection-rate-pruned partial correlation network techniques to facilitate network analysis between the datasets of lipidomic, metabolomic and clinical data. For biomarker identification, a random forest ensemble classification approach was used to both search for valid disease biomarkers and to compare classification performance of lipids, metabolites and clinical factors in combination. Results: We found that steatosis and fibrosis grades were strongly associated with (1) an increase of triglycerides with low carbon number and double bond count as well as (2) a decrease of specific phospholipids, including lysophosphatidylcholines. In addition to the network topology as a result itself, we also present lipid clusters (LCs) of interest to the derived network of proposed interactions in our NAFLD data from the EPoS cohort, along with preliminary metabolite and lipid biomarkers to classify NAFLD fibrosis. Conclusions: Our findings suggest that dysregulation of lipid metabolism in progressive stages of NAFLD is reflected in circulation and may thus hold diagnostic value as well as offer new insights about NAFLD pathogenesis. Using this cohort as a proof-of-concept, we demonstrate current progress in tuning the accuracy random forest approaches with a view to predicting various subtypes of NAFLD patient using a minimal set of lipidomic and metabolic markers. For the first time, a detailed network-based picture emerges between lipids, polar metabolites and clinical variables. Lipidomic / metabolomic markers may provide an alternative method of NAFLD patient classification and risk stratification to guide therapy.
Institute:Örebro University
Last Name:McGlinchey
First Name:Aidan
Address:School of Medical Sciences, Örebro, Örebro, 70281, Sweden
Email:aidan.mcglinchey@oru.se
Phone:+46736485638

Subject:

Subject ID:SU001787
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 Data type Sex NAFLD.Category T2DM Kleiner.Steatosis Inflammation
SA1590861022385746Serum lipidomics F 1 NA 1 -
SA1590871022385747Serum lipidomics F 1 NA 1 1
SA1590881022385761Serum lipidomics F 1 NA 1 1
SA1590891022385792Serum lipidomics F 1 NA 1 3
SA1590901022385834Serum lipidomics F 1 NA 2 1
SA1590911022385816Serum lipidomics F 1 NA 2 1
SA1590921022385771Serum lipidomics F 1 NA 2 2
SA1590931022385802Serum lipidomics F 1 NA 3 1
SA1590941022385801Serum lipidomics F 1 NA 3 1
SA1590951022385768Serum lipidomics F 1 NA 3 1
SA1590961022385786Serum lipidomics F 1 NA 3 2
SA1590971022385784Serum lipidomics F 1 NA 3 2
SA1590981022385755Serum lipidomics F 1 NA 3 2
SA1590991022385754Serum lipidomics F 1 NA 3 2
SA1591001022385832Serum lipidomics F 1 NA 3 2
SA1591011022385865Serum lipidomics F 1 N 1 -
SA1591021022386491Serum lipidomics F 1 N 1 -
SA1591031028971690Serum lipidomics F 1 N 1 -
SA1591041028940127Serum lipidomics F 1 N 1 -
SA1591051022386174Serum lipidomics F 1 N 1 -
SA1591061026694081Serum lipidomics F 1 N 1 -
SA1591071022385897Serum lipidomics F 1 N 1 1
SA1591081022386559Serum lipidomics F 1 N 1 1
SA1591091022385762Serum lipidomics F 1 N 1 1
SA1591101022385836Serum lipidomics F 1 N 1 1
SA1591111022386512Serum lipidomics F 1 N 1 1
SA1591121022386514Serum lipidomics F 1 N 1 1
SA1591131022386602Serum lipidomics F 1 N 1 1
SA1591141022385872Serum lipidomics F 1 N 1 1
SA1591151022385880Serum lipidomics F 1 N 1 1
SA1591161028939921Serum lipidomics F 1 N 1 1
SA1591171022385912Serum lipidomics F 1 N 1 1
SA1591181022385905Serum lipidomics F 1 N 1 1
SA1591191022386990Serum lipidomics F 1 N 1 1
SA1591201022386190Serum lipidomics F 1 N 1 1
SA1591211022386422Serum lipidomics F 1 N 1 1
SA1591221028938105Serum lipidomics F 1 N 1 2
SA1591231022386925Serum lipidomics F 1 N 2 -
SA1591241022386548Serum lipidomics F 1 N 2 -
SA1591251022386196Serum lipidomics F 1 N 2 -
SA1591261022386594Serum lipidomics F 1 N 2 -
SA1591271026694239Serum lipidomics F 1 N 2 1
SA1591281022386943Serum lipidomics F 1 N 2 1
SA1591291022386572Serum lipidomics F 1 N 2 1
SA1591301022385839Serum lipidomics F 1 N 2 2
SA1591311028938200Serum lipidomics F 1 N 2 2
SA1591321022386199Serum lipidomics F 1 N 2 2
SA1591331022386586Serum lipidomics F 1 N 2 3
SA1591341022385777Serum lipidomics F 1 N 3 1
SA1591351026698295Serum lipidomics F 1 N 3 1
SA1591361022386494Serum lipidomics F 1 N 3 2
SA1591371022385780Serum lipidomics F 1 N 3 2
SA1591381026697459Serum lipidomics F 1 N 3 2
SA1591391022386537Serum lipidomics F 1 N 3 2
SA1591401022386517Serum lipidomics F 1 N NA 1
SA1591411028938307Serum lipidomics F 1 N NA NA
SA1591431022386450Serum lipidomics F 1 Y 1 -
SA1591441028971722Serum lipidomics F 1 Y 1 -
SA1591451028938157Serum lipidomics F 1 Y 1 1
SA1591461022386593Serum lipidomics F 1 Y 1 1
SA1591471022386206Serum lipidomics F 1 Y 1 1
SA1591481022386493Serum lipidomics F 1 Y 1 1
SA1591491028939905Serum lipidomics F 1 Y 1 1
SA1591501022385758Serum lipidomics F 1 Y 3 -
SA1591511022386466Serum lipidomics F 1 Y 3 -
SA1591521022386583Serum lipidomics F 1 Y 3 1
SA1591531022385807Serum lipidomics F 1 Y 3 1
SA1591421022386482Serum lipidomics F 1 Y - 2
SA1591541022385803Serum lipidomics F 2 NA 2 2
SA1591551022385810Serum lipidomics F 2 NA 2 2
SA1591561022385745Serum lipidomics F 2 NA 3 1
SA1591571022385770Serum lipidomics F 2 NA 3 3
SA1591581028939780Serum lipidomics F 2 N 1 -
SA1591591022386150Serum lipidomics F 2 N 1 -
SA1591601022386903Serum lipidomics F 2 N 1 1
SA1591611022386454Serum lipidomics F 2 N 1 1
SA1591621028938244Serum lipidomics F 2 N 1 1
SA1591631022386991Serum lipidomics F 2 N 1 1
SA1591641022387178Serum lipidomics F 2 N 1 1
SA1591651028939875Serum lipidomics F 2 N 1 1
SA1591661022386576Serum lipidomics F 2 N 1 1
SA1591671028938158Serum lipidomics F 2 N 1 1
SA1591681022386498Serum lipidomics F 2 N 1 2
SA1591691022385924Serum lipidomics F 2 N 1 2
SA1591701022385859Serum lipidomics F 2 N 1 2
SA1591711022386131Serum lipidomics F 2 N 1 2
SA1591721022386478Serum lipidomics F 2 N 2 -
SA1591731022387102Serum lipidomics F 2 N 2 1
SA1591741028971662Serum lipidomics F 2 N 2 1
SA1591751022386561Serum lipidomics F 2 N 2 1
SA1591761022387123Serum lipidomics F 2 N 2 1
SA1591771022385845Serum lipidomics F 2 N 2 1
SA1591781022386912Serum lipidomics F 2 N 2 1
SA1591791028971709Serum lipidomics F 2 N 2 1
SA1591801022385892Serum lipidomics F 2 N 2 1
SA1591811022386181Serum lipidomics F 2 N 2 1
SA1591821022387151Serum lipidomics F 2 N 2 2
SA1591831022386557Serum lipidomics F 2 N 2 2
SA1591841022387205Serum lipidomics F 2 N 2 2
SA1591851022386163Serum lipidomics F 2 N 2 2
Showing page 1 of 7     Results:    1  2  3  4  5  Next  Last     Showing results 1 to 100 of 627

Collection:

Collection ID:CO001780
Collection Summary:Serum samples were randomized and extracted using a modified version of the previously-published Folch procedure, as applied recently. The maternal samples were analysed as one batch and the cord blood samples as a second batch. In short, 10 µL of 0.9% NaCl and, 120 µL of CHCl3: MeOH (2:1, v/v) containing the internal standards (c = 2.5 µg/mL) was added to 10 µL of each serum sample. The standard solution contained the following compounds: 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (PE(17:0/17:0)), N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (SM(d18:1/17:0)), N-heptadecanoyl-D-erythro-sphingosine (Cer(d18:1/17:0)), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC(17:0/17:0)), 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(17:0)) and 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (PC(16:0/d31/18:1)), were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and, triheptadecanoylglycerol (TG(17:0/17:0/17:0)) was purchased from Larodan AB (Solna, Sweden). The samples were vortex mixed and incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min). 60 µL from the lower layer of each sample was then transferred to a glass vial with an insert and 60 µL of CHCl3: MeOH (2:1, v/v) was added to each sample. The samples were stored at -80 °C until analysis. Calibration curves using 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(16:0e/18:1(9Z))), 1-(1Z-octadecenyl)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(18:0p/18:1(9Z))), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:0)), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:1)), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)), 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PC(18:0p/22:6)) and 1-stearoyl-2-linoleoyl-sn-glycerol (DG(18:0/18:2)), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (LPE(18:1)), N-(9Z-octadecenoyl)-sphinganine (Cer(d18:0/18:1(9Z))), 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)) from Avanti Polar Lipids, 1-Palmitoyl-2-Hydroxy-sn-Glycero-3-Phosphatidylcholine (LPC(16:0)), 1,2,3 trihexadecanoalglycerol (TG(16:0/16:0/16:0)), 1,2,3-trioctadecanoylglycerol (TG(18:0/18:0/18:)) and 3β-hydroxy-5-cholestene-3-stearate (ChoE(18:0)), 3β-Hydroxy-5-cholestene-3-linoleate (ChoE(18:2)) from Larodan, were prepared to the following concentration levels: 100, 500, 1000, 1500, 2000 and 2500 ng/mL (in CHCl3:MeOH, 2:1, v/v) including 1250 ng/mL of each internal standard. The samples were analyzed by ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS). Briefly, the UHPLC system used in this work was a 1290 Infinity II system from Agilent Technologies (Santa Clara, CA, USA). The system was equipped with a multi sampler (maintained at 10 °C), a quaternary solvent manager and a column thermostat (maintained at 50 °C). Injection volume was 1 µL and the separations were performed on an ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm) by Waters (Milford, MA, USA). The mass spectrometer coupled to the UHPLC was a 6545 QTOF from Agilent Technologies interfaced with a dual jet stream electrospray (Ddual ESI) ion source. All analyses were performed in positive ion mode and MassHunter B.06.01 (Agilent Technologies) was used for all data acquisition. Quality control was performed throughout the dataset by including blanks, pure standard samples, extracted standard samples and control serum samples, including in-house serum and a pooled QC with an aliquot of each sample was collected and pooled and used as quality control sample. Relative standard deviations (% RSDs) for identified lipids in the control serum samples (n = 13) and in the pooled serum samples (n = 54) were on average 22.4% and 17.5%, respectively. Mass spectrometry data processing was performed using the open source software package MZmine 2.18. The following steps were applied in this processing: (i) Crop filtering with a m/z range of 350 – 1200 m/z and an RT range of 2.0 to 12 minutes, (ii) Mass detection with a noise level of 750, (iii) Chromatogram builder with a minimum time span of 0.08 min, minimum height of 1000 and a m/z tolerance of 0.006 m/z or 10.0 ppm, (iv) Chromatogram deconvolution using the local minimum search algorithm with a 70% chromatographic threshold, 0.05 min minimum RT range, 5% minimum relative height, 1200 minimum absolute height, a minimum ration of peak top/edge of 1.2 and a peak duration range of 0.08 - 5.0, (v), Isotopic peak grouper with a m/z tolerance of 5.0 ppm, RT tolerance of 0.05 min, maximum charge of 2 and with the most intense isotope set as the representative isotope, (vi) Peak filter with minimum 12 data points, a FWHM between 0.0 and 0.2, tailing factor between 0.45 and 2.22 and asymmetry factor between 0.40 and 2.50, (vii) Join aligner with a m/z tolerance of 0.009 or 10.0 ppm and a weight for of 2, a RT tolerance of 0.1 min and a weight of 1 and with no requirement of charge state or ID and no comparison of isotope pattern, (viii) Peak list row filter with a minimum of 10% of the samples (ix) Gap filling using the same RT and m/z range gap filler algorithm with an m/z tolerance of 0.009 m/z or 11.0 ppm, (x) Identification of lipids using a custom database search with an m/z tolerance of 0.009 m/z or 10.0 ppm and a RT tolerance of 0.1 min, and (xi) Normalization using internal standards PE(17:0/17:0), SM(d18:1/17:0), Cer(d18:1/17:0), LPC(17:0), TG(17:0/17:0/17:0) and PC(16:0/d30/18:1)) for identified lipids and closest ISTD for the unknown lipids followed by calculation of the concentrations based on lipid-class concentration curves.
Sample Type:Blood (serum)

Treatment:

Treatment ID:TR001800
Treatment Summary:No treatment applied.

Sample Preparation:

Sampleprep ID:SP001793
Sampleprep Summary:Serum samples were randomized and extracted using a modified version of the previously-published Folch procedure, as applied recently. The maternal samples were analysed as one batch and the cord blood samples as a second batch. In short, 10 µL of 0.9% NaCl and, 120 µL of CHCl3: MeOH (2:1, v/v) containing the internal standards (c = 2.5 µg/mL) was added to 10 µL of each serum sample. The standard solution contained the following compounds: 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (PE(17:0/17:0)), N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (SM(d18:1/17:0)), N-heptadecanoyl-D-erythro-sphingosine (Cer(d18:1/17:0)), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC(17:0/17:0)), 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(17:0)) and 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (PC(16:0/d31/18:1)), were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and, triheptadecanoylglycerol (TG(17:0/17:0/17:0)) was purchased from Larodan AB (Solna, Sweden). The samples were vortex mixed and incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min). 60 µL from the lower layer of each sample was then transferred to a glass vial with an insert and 60 µL of CHCl3: MeOH (2:1, v/v) was added to each sample. The samples were stored at -80 °C until analysis. Calibration curves using 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(16:0e/18:1(9Z))), 1-(1Z-octadecenyl)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(18:0p/18:1(9Z))), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:0)), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:1)), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)), 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PC(18:0p/22:6)) and 1-stearoyl-2-linoleoyl-sn-glycerol (DG(18:0/18:2)), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (LPE(18:1)), N-(9Z-octadecenoyl)-sphinganine (Cer(d18:0/18:1(9Z))), 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)) from Avanti Polar Lipids, 1-Palmitoyl-2-Hydroxy-sn-Glycero-3-Phosphatidylcholine (LPC(16:0)), 1,2,3 trihexadecanoalglycerol (TG(16:0/16:0/16:0)), 1,2,3-trioctadecanoylglycerol (TG(18:0/18:0/18:)) and 3β-hydroxy-5-cholestene-3-stearate (ChoE(18:0)), 3β-Hydroxy-5-cholestene-3-linoleate (ChoE(18:2)) from Larodan, were prepared to the following concentration levels: 100, 500, 1000, 1500, 2000 and 2500 ng/mL (in CHCl3:MeOH, 2:1, v/v) including 1250 ng/mL of each internal standard. The samples were analyzed by ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS). Briefly, the UHPLC system used in this work was a 1290 Infinity II system from Agilent Technologies (Santa Clara, CA, USA). The system was equipped with a multi sampler (maintained at 10 °C), a quaternary solvent manager and a column thermostat (maintained at 50 °C). Injection volume was 1 µL and the separations were performed on an ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm) by Waters (Milford, MA, USA). The mass spectrometer coupled to the UHPLC was a 6545 QTOF from Agilent Technologies interfaced with a dual jet stream electrospray (Ddual ESI) ion source. All analyses were performed in positive ion mode and MassHunter B.06.01 (Agilent Technologies) was used for all data acquisition. Quality control was performed throughout the dataset by including blanks, pure standard samples, extracted standard samples and control serum samples, including in-house serum and a pooled QC with an aliquot of each sample was collected and pooled and used as quality control sample. Relative standard deviations (% RSDs) for identified lipids in the control serum samples (n = 13) and in the pooled serum samples (n = 54) were on average 22.4% and 17.5%, respectively. Mass spectrometry data processing was performed using the open source software package MZmine 2.18. The following steps were applied in this processing: (i) Crop filtering with a m/z range of 350 – 1200 m/z and an RT range of 2.0 to 12 minutes, (ii) Mass detection with a noise level of 750, (iii) Chromatogram builder with a minimum time span of 0.08 min, minimum height of 1000 and a m/z tolerance of 0.006 m/z or 10.0 ppm, (iv) Chromatogram deconvolution using the local minimum search algorithm with a 70% chromatographic threshold, 0.05 min minimum RT range, 5% minimum relative height, 1200 minimum absolute height, a minimum ration of peak top/edge of 1.2 and a peak duration range of 0.08 - 5.0, (v), Isotopic peak grouper with a m/z tolerance of 5.0 ppm, RT tolerance of 0.05 min, maximum charge of 2 and with the most intense isotope set as the representative isotope, (vi) Peak filter with minimum 12 data points, a FWHM between 0.0 and 0.2, tailing factor between 0.45 and 2.22 and asymmetry factor between 0.40 and 2.50, (vii) Join aligner with a m/z tolerance of 0.009 or 10.0 ppm and a weight for of 2, a RT tolerance of 0.1 min and a weight of 1 and with no requirement of charge state or ID and no comparison of isotope pattern, (viii) Peak list row filter with a minimum of 10% of the samples (ix) Gap filling using the same RT and m/z range gap filler algorithm with an m/z tolerance of 0.009 m/z or 11.0 ppm, (x) Identification of lipids using a custom database search with an m/z tolerance of 0.009 m/z or 10.0 ppm and a RT tolerance of 0.1 min, and (xi) Normalization using internal standards PE(17:0/17:0), SM(d18:1/17:0), Cer(d18:1/17:0), LPC(17:0), TG(17:0/17:0/17:0) and PC(16:0/d30/18:1)) for identified lipids and closest ISTD for the unknown lipids followed by calculation of the concentrations based on lipid-class concentration curves.
Processing Storage Conditions:-80℃
Extract Storage:-80℃

Combined analysis:

Analysis ID AN002785
Analysis type MS
Chromatography type Normal phase
Chromatography system Agilent 1290 Infinity II
Column Waters Acquity BEH C18 (50 x 2.1mm, 1.7 um)
MS Type ESI
MS instrument type QTOF
MS instrument name Agilent 6545 QTOF
Ion Mode POSITIVE
Units log2-transformed autoscaled values

Chromatography:

Chromatography ID:CH002060
Instrument Name:Agilent 1290 Infinity II
Column Name:Waters Acquity BEH C18 (50 x 2.1mm, 1.7 um)
Chromatography Type:Normal phase

MS:

MS ID:MS002581
Analysis ID:AN002785
Instrument Name:Agilent 6545 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:All analyses were performed in positive ion mode and MassHunter B.06.01 (Agilent Technologies) was used for all data acquisition. Quality control was performed throughout the dataset by including blanks, pure standard samples, extracted standard samples and control serum samples, including in-house serum and a pooled QC with an aliquot of each sample was collected and pooled and used as quality control sample. Relative standard deviations (% RSDs) for identified lipids in the control serum samples (n = 13) and in the pooled serum samples (n = 54) were on average 22.4% and 17.5%, respectively. Regarding data processing, and missing values (likely below limit of detection) were imputed as half of the minimum observed value for that feature. Values were then log2-transformed and subsequently autoscaled (scaled to mean and unit variance).
Ion Mode:POSITIVE
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