Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA096563

Local Sample ID:	20190510_BJJ_Lipids_Sample_3_HB101
Subject ID:	SU001406
Subject Type:	Invertebrate
Subject Species:	Caenorhabditis elegans
Taxonomy ID:	6239

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Subject:

Subject ID:	SU001406
Subject Type:	Invertebrate
Subject Species:	Caenorhabditis elegans
Taxonomy ID:	6239

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
20190510_BJJ_Lipids_Sample_3_HB101	SA096563	FL013622	HB101	Treatment

Collection:

Collection ID:	CO001401
Collection Summary:	LCMS lipid and acyl-carnitine analysis.
Sample Type:	C. elegans

Treatment:

Treatment ID:	TR001421
Treatment Summary:	Parental exposure to environmental stress can program adaptive changes in offspring in diverse organisms (1–4). The mechanisms by which parental exposure to environmental stresses can program predictive adaptive responses in offspring remain almost completely unknown. Here we report that the soil bacteria Pseudomonas vranovensis is a natural pathogen of the nematode Caenorhabditis elegans and that parental exposure of C. elegans to P. vranovensis promotes offspring resistance to infection. This adaptation can be transmitted transgenerationally such that infection of adults can enhance the immunity of their descendants four generations later. We find that parental infection by P. vranovensis results in increased expression of the cysteine synthases CYSL-1 and CYSL-2 and the regulator of hypoxia inducible factor RHY-1 in progeny, that the expression of these three genes in offspring is required for adaptation to P. vranovensis, and that the expression of these genes is regulated by the WD40 repeat protein WDR-23.

Treatment ID:

TR001421

Treatment Summary:

Parental exposure to environmental stress can program adaptive changes in offspring in diverse organisms (1–4). The mechanisms by which parental exposure to environmental stresses can program predictive adaptive responses in offspring remain almost completely unknown. Here we report that the soil bacteria Pseudomonas vranovensis is a natural pathogen of the nematode Caenorhabditis elegans and that parental exposure of C. elegans to P. vranovensis promotes offspring resistance to infection. This adaptation can be transmitted transgenerationally such that infection of adults can enhance the immunity of their descendants four generations later. We find that parental infection by P. vranovensis results in increased expression of the cysteine synthases CYSL-1 and CYSL-2 and the regulator of hypoxia inducible factor RHY-1 in progeny, that the expression of these three genes in offspring is required for adaptation to P. vranovensis, and that the expression of these genes is regulated by the WD40 repeat protein WDR-23.

Sample Preparation:

Sampleprep ID:	SP001414
Sampleprep Summary:	C. elegans was prepared for LC-MS lipidomics and acyl-carnitine analysis as previously described (3) with minor modifications. Briefly, ~40 µL of concentrated embryos were re-suspended in 100 µL of water, then 0.4 mL of chloroform was added to each sample followed by 0.2 mL of methanol containing the stable isotope labelled acyl-carnitine internal standards (Butyryl-L-carnitine-d7 at 5 µM and Hexadecanoyl-L-carnitine-d3 at 5 µM). The samples were then homogenised by vortexing then transferred into a 2 mL Eppendorf screw-cap tube. The original container was washed out with 0.5 mL of chloroform: methanol (2: 1, respectively) and added to the appropriate 2 mL Eppendorf screw-cap tube. This was followed by the addition of 150 µL of the following stable isotope labelled internal standards (approximately 10 to 50 µM in methanol): Ceramide_C16d31, LPC_(C14:0d42), PC_(C16:0d31 / C18:1), PE_(C16:0d31 / C18:1), PG_(C16:0d31 / C18:1), PI_(C16:0d31 / C18:1), PS_(C16:0d62), SM_(C16:0d31), TG_(45:0d29) and TG_(48:0d31). Then, 400 µL of sterile water was added. The samples were vortexed for 1 min, and then centrifuged at ~20,000 rpm for 5 minutes. For the intact lipid sample preparation, 0.3 mL of the organic layer (the lower chloroform layer) was collected into a 2 mL amber glass vial (Agilent Technologies, Santa Clara California, USA) and dried down to dryness in an Eppendorf Concentrator Plus system (Eppendorf, Stevenage, UK) run for 60 minutes at 45 °C. The dried lipid samples were then reconstituted with 100 µL of 2:1:1 solution of propan-2-ol, acetonitrile and water, respectively, and then vortexed thoroughly. The lipid samples were then transferred into a 300 μL low-volume vial insert inside a 2 mL amber glass auto-sample vial ready for liquid chromatography separation with mass spectrometry detection (LC-MS) of intact lipid species. For the acyl-carnitine sample preparation, 0.2 mL of the organic layer (the lower chloroform layer) and 0.2 mL of the aqueous layer (the top water layer) were mixed into a 2 mL amber glass vial and dried down to dryness. The dried acyl-carnitine samples were then reconstituted with 100 µL of water and acetonitrile (4: 1, respectively) and thoroughly vortexed. The acyl-carnitine samples were then transferred into a 300 μL low-volume vial insert inside a 2 mL amber glass auto-sample vial ready for liquid chromatography separation with mass spectrometry detection (LC-MS) of the acyl-carnitine species.

Sampleprep ID:

SP001414

Sampleprep Summary:

C. elegans was prepared for LC-MS lipidomics and acyl-carnitine analysis as previously described (3) with minor modifications. Briefly, ~40 µL of concentrated embryos were re-suspended in 100 µL of water, then 0.4 mL of chloroform was added to each sample followed by 0.2 mL of methanol containing the stable isotope labelled acyl-carnitine internal standards (Butyryl-L-carnitine-d7 at 5 µM and Hexadecanoyl-L-carnitine-d3 at 5 µM). The samples were then homogenised by vortexing then transferred into a 2 mL Eppendorf screw-cap tube. The original container was washed out with 0.5 mL of chloroform: methanol (2: 1, respectively) and added to the appropriate 2 mL Eppendorf screw-cap tube. This was followed by the addition of 150 µL of the following stable isotope labelled internal standards (approximately 10 to 50 µM in methanol): Ceramide_C16d31, LPC_(C14:0d42), PC_(C16:0d31 / C18:1), PE_(C16:0d31 / C18:1), PG_(C16:0d31 / C18:1), PI_(C16:0d31 / C18:1), PS_(C16:0d62), SM_(C16:0d31), TG_(45:0d29) and TG_(48:0d31). Then, 400 µL of sterile water was added. The samples were vortexed for 1 min, and then centrifuged at ~20,000 rpm for 5 minutes. For the intact lipid sample preparation, 0.3 mL of the organic layer (the lower chloroform layer) was collected into a 2 mL amber glass vial (Agilent Technologies, Santa Clara California, USA) and dried down to dryness in an Eppendorf Concentrator Plus system (Eppendorf, Stevenage, UK) run for 60 minutes at 45 °C. The dried lipid samples were then reconstituted with 100 µL of 2:1:1 solution of propan-2-ol, acetonitrile and water, respectively, and then vortexed thoroughly. The lipid samples were then transferred into a 300 μL low-volume vial insert inside a 2 mL amber glass auto-sample vial ready for liquid chromatography separation with mass spectrometry detection (LC-MS) of intact lipid species. For the acyl-carnitine sample preparation, 0.2 mL of the organic layer (the lower chloroform layer) and 0.2 mL of the aqueous layer (the top water layer) were mixed into a 2 mL amber glass vial and dried down to dryness. The dried acyl-carnitine samples were then reconstituted with 100 µL of water and acetonitrile (4: 1, respectively) and thoroughly vortexed. The acyl-carnitine samples were then transferred into a 300 μL low-volume vial insert inside a 2 mL amber glass auto-sample vial ready for liquid chromatography separation with mass spectrometry detection (LC-MS) of the acyl-carnitine species.

Combined analysis:

Analysis ID	AN002221
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Shimadzu 20AD
Column	Waters Acquity UPLC CSH C18(50 x 2.1mm ,1.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Exactive
Ion Mode	UNSPECIFIED
Units	Area ratio

Chromatography:

Chromatography ID:	CH001629
Chromatography Summary:	Full chromatographic separation of intact lipids (4) was achieved using Shimadzu HPLC System (Shimadzu UK Limited, Milton Keynes, United Kingdom) with the injection of 10 µL onto a Waters Acquity UPLC® CSH C18 column; 1.7 µm, I.D. 2.1 mm X 50 mm, maintained at 55 °C. Mobile phase A was 6:4, acetonitrile and water with 10 mM ammonium formate. Mobile phase B was 9:1, propan-2-ol and acetonitrile with 10 mM ammonium formate. The flow was maintained at 500 µL per minute through the following gradient: 0.00 minutes_40% mobile phase B; 0.40 minutes_43% mobile phase B; 0.45 minutes_50% mobile phase B; 2.40 minutes_54% mobile phase B; 2.45 minutes_70% mobile phase B; 7.00 minutes_99% mobile phase B; 8.00 minutes_99% mobile phase B; 8.3 minutes_40% mobile phase B; 10 minutes_40% mobile phase B; 10.00 minutes_40% mobile phase B. The sample injection needle was washed using 9:1, 2-propan-2-ol and acetonitrile with 0.1 % formic acid. The mass spectrometer used was the Thermo Scientific Exactive Orbitrap with a heated electrospray ionisation source (Thermo Fisher Scientific, Hemel Hempstead, UK). The mass spectrometer was calibrated immediately before sample analysis using positive and negative ionisation calibration solution (recommended by Thermo Scientific). Additionally, the heated electrospray ionisation source was optimised at 50:50 mobile phase A to mobile phase B for spray stability (capillary temperature; 380 °C, source heater temperature; 420 °C, sheath gas flow; 60 (arbitrary), auxiliary gas flow; 20 (arbitrary), sweep gas; 5 (arbitrary), source voltage; 3.5 kV. The mass spectrometer resolution was set to 25,000 with a full-scan range of m/z 100 to 1,800 Da, with continuous switching between positive and negative mode. Lipid quantification was achieved using the area under the curve (AUC) of the corresponding high resolution extracted ion chromatogram (with a window of ± 8 ppm) at the indicative retention time. The lipid analyte AUC relative to the associated internal standard AUC for that lipid class was used to semi-quantify and correct for any extraction/instrument variation.
Instrument Name:	Shimadzu 20AD
Column Name:	Waters Acquity UPLC CSH C18(50 x 2.1mm ,1.7um)
Column Temperature:	55
Flow Gradient:	0.00 minutes_40% mobile phase B; 0.40 minutes_43% mobile phase B; 0.45 minutes_50% mobile phase B; 2.40 minutes_54% mobile phase B; 2.45 minutes_70% mobile phase B; 7.00 minutes_99% mobile phase B; 8.00 minutes_99% mobile phase B; 8.3 minutes_40% mobile phase B; 10 minutes_40% mobile phase B; 10.00 minutes_40% mobile phase B. The sample injection needle was washed using 9:1, 2-propan-2-ol and acetonitrile with 0.1 % formic acid.
Flow Rate:	500 µL/min
Solvent A:	60% acetonitrile/40% water; 10 mM ammonium formate
Solvent B:	90% isopropanol/10% acetonitrile; 10 mM ammonium formate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002067
Analysis ID:	AN002221
Instrument Name:	Thermo Exactive
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The mass spectrometer used was the Thermo Scientific Exactive Orbitrap with a heated electrospray ionisation source (Thermo Fisher Scientific, Hemel Hempstead, UK). The mass spectrometer was calibrated immediately before sample analysis using positive and negative ionisation calibration solution (recommended by Thermo Scientific). Additionally, the heated electrospray ionisation source was optimised at 50:50 mobile phase A to mobile phase B for spray stability (capillary temperature; 380 °C, source heater temperature; 420 °C, sheath gas flow; 60 (arbitrary), auxiliary gas flow; 20 (arbitrary), sweep gas; 5 (arbitrary), source voltage; 3.5 kV. The mass spectrometer resolution was set to 25,000 with a full-scan range of m/z 100 to 1,800 Da, with continuous switching between positive and negative mode. Lipid quantification was achieved using the area under the curve (AUC) of the corresponding high resolution extracted ion chromatogram (with a window of ± 8 ppm) at the indicative retention time. The lipid analyte AUC relative to the associated internal standard AUC for that lipid class was used to semi-quantify and correct for any extraction/instrument variation.
Ion Mode:	UNSPECIFIED