Summary of Study ST003326

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 PR002068. The data can be accessed directly via it's Project DOI: 10.21228/M89F9K This work is supported by NIH grant, U2C- DK119886.

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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 IDST003326
Study TitleLipidome profiling in non-alcoholic steatohepatitis identifies phosphatidylserine synthase 1 as a regulator of hepatic lipoprotein metabolism
Study SummaryNon-alcoholic fatty liver disease and more progressive non-alcoholic steatohepatitis (NASH) are characterized by defective lipid metabolism, which causes hepatic steatosis and disease progression. However, the changes in lipid metabolism in NASH are incompletely understood. Using lipidome profiling in livers of eight mouse strains, that differ substantially in susceptibility to NASH and liver fibrosis, as well as in patients with NASH, we show that phosphatidylserine (PS) accumulation and preservation of PS synthase 1 (PSS1) expression is associated with resistance to NASH. Mechanistically, PSS1 overexpression in the liver reduces hepatic steatosis through remodeling of the hepatic and liver-derived VLDL lipidome in mice with NASH. Specifically, we show an increase in VLDL ceramide content that suppresses the expression and activity of lipoprotein lipase (LPL) in skeletal muscle, thereby reducing VLDL-triglyceride clearance, fatty acid uptake and lipid accumulation in skeletal muscle. In addition, remodelling of lipoprotein composition inhibits the LDL receptor in the liver, likely contributing to the reduction in hepatic steatosis. Together, this study provides a unique resource describing lipidome changes in NASH, and identifies PSS1 as a novel regulator of hepatic lipoprotein metabolism.
Institute
University of Melbourne
Last NameMontgomery
First NameMagdalene
AddressCorner Grattan Street & Royal Parade
Emailmagdalene.montgomery@unimelb.edu.au
Phone0422059907
Submit Date2024-05-13
Raw Data AvailableYes
Raw Data File Type(s)abf
Analysis Type DetailLC-MS
Release Date2024-10-21
Release Version1
Magdalene Montgomery Magdalene Montgomery
https://dx.doi.org/10.21228/M89F9K
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002068
Project DOI:doi: 10.21228/M89F9K
Project Title:Lipidome profiling in non-alcoholic steatohepatitis identifies phosphatidylserine synthase 1 as a regulator of hepatic lipoprotein metabolism
Project Summary:This project aimed to investigate the regulation of the hepatic lipidome in mice with non-alcoholic steatohepatitis (NASH) and liver fibrosis, and assess the differences in the hepatic lipidome in mouse strains susceptible or resistant to NASH and metabolic comorbidities. For this purpose, eight mouse strains were fed either a chow control diet or a western-style diet enriched in lipid, carbohydrate and cholesterol for 30-32 weeks, followed by in-depth metabolic phenotyping, assessment of liver pathology and the hepatic lipidome by LC/MS.  This study found that resistance to diet-induced NASH in BALB/c mice was associated with increased hepatic phosphatidylserine (PS) content. Subsequent studies focusing on overexpression of PS synthase 1 (PSS1) in the livers of mice with steatosis or NASH show that increasing hepatic PS is associated with improvements in steatosis and/or inflammation.
Institute:University of Melbourne
Last Name:Montgomery
First Name:Magdalene
Address:Corner Grattan Street Royal Parade, Melbourne, Victoria, 3010, Australia
Email:magdalene.montgomery@unimelb.edu.au
Phone:0422059907

Subject:

Subject ID:SU003447
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:A/J, BALB/c, C3H/HeJ, C57BL/6J, CBA/CaH, DBA/2J, FVB/NJ and NOD/ShiLtJ mice
Age Or Age Range:Mice were fed a low-fat or western-style diet for 30-32 weeks
Weight Or Weight Range:30-45 grams
Gender:Male
Animal Animal Supplier:Animal Resources Centre, Canning Vale, Australia
Animal Housing:Group housing (3-5 mice/cage)
Animal Light Cycle:12/12 light/dark
Animal Feed:low-fat chow or high-fat high-sugar high-cholesterol diet (SF16-033 Specialty Feeds, Australia)
Animal Water:ad libitum water

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Mouse Strain Food source Sample source
SA361345210226_Hamze_MM-1835_055pA/J Chow Liver
SA361346210226_Hamze_MM-1835_051nA/J Chow Liver
SA361347210226_Hamze_MM-1835_053nA/J Chow Liver
SA361348210226_Hamze_MM-1835_055nA/J Chow Liver
SA361349210226_Hamze_MM-1835_057nA/J Chow Liver
SA361350210226_Hamze_MM-1835_059nA/J Chow Liver
SA361351210226_Hamze_MM-1835_061nA/J Chow Liver
SA361352210226_Hamze_MM-1835_063nA/J Chow Liver
SA361353210226_Hamze_MM-1835_065nA/J Chow Liver
SA361354210226_Hamze_MM-1835_067nA/J Chow Liver
SA361355210226_Hamze_MM-1835_068nA/J Chow Liver
SA361356210226_Hamze_MM-1835_053pA/J Chow Liver
SA361357210226_Hamze_MM-1835_051pA/J Chow Liver
SA361358210226_Hamze_MM-1835_068pA/J Chow Liver
SA361359210226_Hamze_MM-1835_067pA/J Chow Liver
SA361360210226_Hamze_MM-1835_059pA/J Chow Liver
SA361361210226_Hamze_MM-1835_061pA/J Chow Liver
SA361362210226_Hamze_MM-1835_065pA/J Chow Liver
SA361363210226_Hamze_MM-1835_057pA/J Chow Liver
SA361364210226_Hamze_MM-1835_063pA/J Chow Liver
SA361365210226_Hamze_MM-1835_056pA/J NASH Liver
SA361366210226_Hamze_MM-1835_064pA/J NASH Liver
SA361367210226_Hamze_MM-1835_052nA/J NASH Liver
SA361368210226_Hamze_MM-1835_054nA/J NASH Liver
SA361369210226_Hamze_MM-1835_066pA/J NASH Liver
SA361370210226_Hamze_MM-1835_056nA/J NASH Liver
SA361371210226_Hamze_MM-1835_058nA/J NASH Liver
SA361372210226_Hamze_MM-1835_062nA/J NASH Liver
SA361373210226_Hamze_MM-1835_060nA/J NASH Liver
SA361374210226_Hamze_MM-1835_062pA/J NASH Liver
SA361375210226_Hamze_MM-1835_064nA/J NASH Liver
SA361376210226_Hamze_MM-1835_066nA/J NASH Liver
SA361377210226_Hamze_MM-1835_060pA/J NASH Liver
SA361378210226_Hamze_MM-1835_052pA/J NASH Liver
SA361379210226_Hamze_MM-1835_058pA/J NASH Liver
SA361380210226_Hamze_MM-1835_054pA/J NASH Liver
SA361381210226_Hamze_MM-1835_030pBALBc Chow Liver
SA361382210226_Hamze_MM-1835_029pBALBc Chow Liver
SA361383210226_Hamze_MM-1835_034pBALBc Chow Liver
SA361384210226_Hamze_MM-1835_033pBALBc Chow Liver
SA361385210226_Hamze_MM-1835_037pBALBc Chow Liver
SA361386210226_Hamze_MM-1835_029nBALBc Chow Liver
SA361387210226_Hamze_MM-1835_030nBALBc Chow Liver
SA361388210226_Hamze_MM-1835_033nBALBc Chow Liver
SA361389210226_Hamze_MM-1835_034nBALBc Chow Liver
SA361390210226_Hamze_MM-1835_035nBALBc Chow Liver
SA361391210226_Hamze_MM-1835_036nBALBc Chow Liver
SA361392210226_Hamze_MM-1835_037nBALBc Chow Liver
SA361393210226_Hamze_MM-1835_036pBALBc Chow Liver
SA361394210226_Hamze_MM-1835_035pBALBc Chow Liver
SA361395210226_Hamze_MM-1835_026nBALBc NASH Liver
SA361396210226_Hamze_MM-1835_024nBALBc NASH Liver
SA361397210226_Hamze_MM-1835_023nBALBc NASH Liver
SA361398210226_Hamze_MM-1835_022nBALBc NASH Liver
SA361399210226_Hamze_MM-1835_021nBALBc NASH Liver
SA361400210226_Hamze_MM-1835_022pBALBc NASH Liver
SA361401210226_Hamze_MM-1835_021pBALBc NASH Liver
SA361402210226_Hamze_MM-1835_025nBALBc NASH Liver
SA361403210226_Hamze_MM-1835_028nBALBc NASH Liver
SA361404210226_Hamze_MM-1835_027nBALBc NASH Liver
SA361405210226_Hamze_MM-1835_023pBALBc NASH Liver
SA361406210226_Hamze_MM-1835_024pBALBc NASH Liver
SA361407210226_Hamze_MM-1835_025pBALBc NASH Liver
SA361408210226_Hamze_MM-1835_026pBALBc NASH Liver
SA361409210226_Hamze_MM-1835_027pBALBc NASH Liver
SA361410210226_Hamze_MM-1835_028pBALBc NASH Liver
SA361411210226_Hamze_MM-1835_006pBL6 Chow Liver
SA361412210226_Hamze_MM-1835_003pBL6 Chow Liver
SA361413210226_Hamze_MM-1835_004pBL6 Chow Liver
SA361414210226_Hamze_MM-1835_005pBL6 Chow Liver
SA361415210226_Hamze_MM-1835_010pBL6 Chow Liver
SA361416210226_Hamze_MM-1835_007pBL6 Chow Liver
SA361417210226_Hamze_MM-1835_008pBL6 Chow Liver
SA361418210226_Hamze_MM-1835_009pBL6 Chow Liver
SA361419210226_Hamze_MM-1835_002nBL6 Chow Liver
SA361420210226_Hamze_MM-1835_002pBL6 Chow Liver
SA361421210226_Hamze_MM-1835_001nBL6 Chow Liver
SA361422210226_Hamze_MM-1835_001pBL6 Chow Liver
SA361423210226_Hamze_MM-1835_007nBL6 Chow Liver
SA361424210226_Hamze_MM-1835_009nBL6 Chow Liver
SA361425210226_Hamze_MM-1835_008nBL6 Chow Liver
SA361426210226_Hamze_MM-1835_010nBL6 Chow Liver
SA361427210226_Hamze_MM-1835_006nBL6 Chow Liver
SA361428210226_Hamze_MM-1835_005nBL6 Chow Liver
SA361429210226_Hamze_MM-1835_004nBL6 Chow Liver
SA361430210226_Hamze_MM-1835_003nBL6 Chow Liver
SA361431210226_Hamze_MM-1835_011pBL6 NASH Liver
SA361432210226_Hamze_MM-1835_013pBL6 NASH Liver
SA361433210226_Hamze_MM-1835_014pBL6 NASH Liver
SA361434210226_Hamze_MM-1835_015pBL6 NASH Liver
SA361435210226_Hamze_MM-1835_016pBL6 NASH Liver
SA361436210226_Hamze_MM-1835_017pBL6 NASH Liver
SA361437210226_Hamze_MM-1835_018pBL6 NASH Liver
SA361438210226_Hamze_MM-1835_019pBL6 NASH Liver
SA361439210226_Hamze_MM-1835_020pBL6 NASH Liver
SA361440210226_Hamze_MM-1835_012pBL6 NASH Liver
SA361441210226_Hamze_MM-1835_020nBL6 NASH Liver
SA361442210226_Hamze_MM-1835_011nBL6 NASH Liver
SA361443210226_Hamze_MM-1835_012nBL6 NASH Liver
SA361444210226_Hamze_MM-1835_013nBL6 NASH Liver
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Collection:

Collection ID:CO003440
Collection Summary:This collection contains data on the liver lipidome across 8 inbred mouse strains fed either a chow control diet or a western-style diet enriched in lipid, carbohydrate and cholesterol. Untargeted lipidomics data available from 144 mice across eight mouse strains and two dietary regimes.
Sample Type:Liver

Treatment:

Treatment ID:TR003456
Treatment Summary:For the assessment of mouse strain-dependent variation in the hepatic lipidome, eight-week-old male A/J, BALB/c, C3H/HeJ, C57BL/6J, CBA/CaH, DBA/2J, FVB/NJ and NOD/ShiLtJ mice (Animal Resources Centre, Canning Vale, Australia) were fed either a rodent chow diet (5% energy from fat) or a diet enriched in fat (40% energy from lard), carbohydrate (40% energy from carbohydrates, of this 20% fructose), and 2% cholesterol (SF16-033 Specialty Feeds, Australia) for a total of 30-32 weeks (referred to as NASH diet).

Sample Preparation:

Sampleprep ID:SP003454
Sampleprep Summary:Lipids from whole liver were extracted using a monophasic extraction protocol. Briefly, 5-10 mg of liver was homogenized using a Precellys tissue homogenizer in 100 µL 1:1 butanol-methanol, containing 5 µL of SPLASH® II LIPIDOMIX® Mass Spec Standard (part no. 330709W, Avanti Polar Lipids Inc) and 5 uL of Ceramide LIPIDOMIX® Mass Spec Standard (part no. 330712X, Avanti Polar Lipids Inc). Samples were mixed for 1 h at room temperature, centrifuged (14,000 g, 10 min, 20 ⁰C) and transferred into sample vials with glass inserts for analysis.

Combined analysis:

Analysis ID AN005448 AN005449
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Agilent ZORBAX Eclipse Plus C18 (100 x 2.1mm,1.8um) Agilent ZORBAX Eclipse Plus C18 (100 x 2.1mm,1.8um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Fusion Orbitrap Thermo Fusion Orbitrap
Ion Mode NEGATIVE POSITIVE
Units pmol/mg tissue pmol/mg tissue

Chromatography:

Chromatography ID:CH004136
Instrument Name:Thermo Vanquish
Column Name:Agilent ZORBAX Eclipse Plus C18 (100 x 2.1mm,1.8um)
Column Temperature:60
Flow Gradient:a flow rate of 350 uL/min for 3 min using 30% solvent B. During separation, the percentage of solvent B was increased from 30% to 70% in 5 min, from 70% to 93% in 9 min and, from 93% to 99% in 7 min, and from 91% to 97% in 31 min. Subsequently, the percentage of solvent B was increased to 99.5% in 0.1 min and then maintained at 99.5% for 3 min. Finally, the percentage of solvent B was decreased to 30% in 0.1 min and maintained for 3.9 min
Flow Rate:350 uL/min
Solvent A:60% Acetonitrile/40% Water; 10 mM ammonium acetate; 5 uM medronic acid
Solvent B:90% Isopropanol/10% Acetonitrile; 10 mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS005174
Analysis ID:AN005448
Instrument Name:Thermo Fusion Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All experiments were performed using a Heated Electrospray Ionization (HESI) source. The spray voltages were 3.5 kV in positive ionisation-mode and 3.0 kV in negative ionisation-mode. In both polarities, the flow rates of sheath, auxiliary and sweep gases were 20 and 6 and 1 ‘arbitrary’ unit(s), respectively. The ion transfer tube and vaporizer temperatures were maintained at 350 °C and 400 °C, respectively, and the S-Lens RF level was set at 50%. In the positive ionisation-mode from 3 to 24 min, top speed data-dependent scan with a cycle time of 1 s was used. Within each cycle, a full-scan MS spectra were acquired firstly in the Orbitrap at a mass resolving power of 120,000 (at m/z 200) across an m/z range of 300–2000 using quadrupole isolation, an automatic gain control (AGC) target of 4e5 and a maximum injection time of 50 milliseconds, followed by higher-energy collisional dissociation (HCD)-MS/MS at a mass resolving power of 15,000 (at m/z 200), a normalised collision energy (NCE) of 27% at positive mode and 30% at negative mode, an m/z isolation window of 1, a maximum injection time of 22 milliseconds and an AGC target of 5e4. For the improved structural characterisation of glycerophosphocholine (PC) lipid cations, a data-dependent product ion (m/z 184.0733)-triggered collision-induced dissociation (CID)-MS/MS scan was performed in the cycle using a q-value of 0.25 and a NCE of 30%, with other settings being the same as that for HCD-MS/MS. For the improved structural characterisation of triacylglycerol (TG) lipid cations, the fatty acid + NH3 neutral loss product ions observed by HCD-MS/MS were used to trigger the acquisition of the top-3 data-dependent CID-MS3 scans in the cycle using a q-value of 0.25 and a NCE of 30%, with other settings being the same as that for HCD-MS/MS. Identification and quantification of lipids LC-MS/MS data was searched through MS Dial 4.48. The mass accuracy settings are 0.005 Da and 0.025 Da for MS1 and MS2. The minimum peak height is 50000 and mass slice width is 0.05 Da. The identification score cut off is 80%. Post identification was done with a text file containing name and m/z of each standard in SPLASH® II LIPIDOMIX® Mass Spec Standard. In positive mode, [M+H]+, [M+NH4]+ and [M+H-H2O]+ were selected as ion forms and lipid classes including CAR, LPC, LPE, PC, PE, PS, CL, EtherLPC, EtherLPE, EtherPC, EtherPE, Sph, DHSph, SM, MG, DG, EtherDG, TG, EtherTG, CE, CoQ, Cer_NS, Cer_NDS, CerP, HexCer_NS, HexCer_NDS, Hex2Cer, Hex3Cer and ST were selected for the search. In negative mode, [M-H]- and [M+CH3COO]- were selected as ion forms and lipid classes including LPS, LPG, LPI, LPA, PA, PC, PE, PG, PI, PS, CL, EtherPC, EtherPE, EtherPS, EtherPI, EtherPG, EtherLPG, SM, Cer_NS, Cer_NDS, CerP, HexCer_NS, HexCer_NDS, Hex2Cer, Hex3Cer, SHexCer, NAE and GM3 were selected for the search. The retention time tolerance for alignment is 0.1 min. The peak count filter is 50% and the N% detected in at least one group is 66%. Lipids with maximum intensity less than 5-fold of average intensity in blank was removed. All other settings were default. All lipid LC-MS features were manually inspected and re-integrated when needed. These four types of lipids, 1) Cer_NS, Cer_NDS and CL with only sum composition, 2) lipid identification due to peak tailing, 3) retention time outliner within each lipid class, 4) LPA and PA generated by in-source fragmentation of LPS and PS were also removed. Relative quantification of lipid species was achieved by comparison of the LC peak areas of identified lipids against those of the corresponding internal lipid standards in the same lipid class, and the resultant ratio of peak area was then normalized to weight of tissue and total PC content. For the lipid classes without correspondent isotope-labelled internal lipid standards, the LC peak areas of individual molecular species within these classes were normalised as follows: the MG species against the DG (18:1D7_15:0) internal standard; the CL, LPG, PG, LPA and PA against the PI (18:1D7_15:0) internal standard; the LPS against the PS (18:1D7_15:0) internal standard; the Hex1Cer against the SM (d36:2D9) internal standard. Given that only a single lipid standard per class was used, some of the identified lipids were normalised against a standard from a different class or sub-class, and no attempts were made to quantitatively correct for different ESI responses of individual lipids due to concentration, acyl chain length, degree of unsaturation, or matrix effects caused by differences in chromatographic retention times compared with the relevant standards. The results reported here are for relative quantification and should not be considered to reflect the absolute concentrations of each lipid or lipid sub-class. Lipidomics data analysis All lipidomics data were further processed with Perseus (Version 1.5.0.40). Within Perseus, values were log2 transformed, each lipid species normalized to the z-score, and the replicates grouped accordingly. All lipid species that had less than 70 percent of “valid value” in each group were removed and the missing values were replaced by imputation. A two-sample t-test (FDR < 5%) was performed to obtain a list of significantly regulated lipids between diets within each strain. Lastly, lipidomics data were analysed for biophysical data, lipid functions and organelle associations using the Lipid Ontology (LION) enrichment analysis web application, as well as BioPAN (Bioinformatics Methodology For Pathway Analysis).
Ion Mode:NEGATIVE
  
MS ID:MS005175
Analysis ID:AN005449
Instrument Name:Thermo Fusion Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All experiments were performed using a Heated Electrospray Ionization (HESI) source. The spray voltages were 3.5 kV in positive ionisation-mode and 3.0 kV in negative ionisation-mode. In both polarities, the flow rates of sheath, auxiliary and sweep gases were 20 and 6 and 1 ‘arbitrary’ unit(s), respectively. The ion transfer tube and vaporizer temperatures were maintained at 350 °C and 400 °C, respectively, and the S-Lens RF level was set at 50%. In the positive ionisation-mode from 3 to 24 min, top speed data-dependent scan with a cycle time of 1 s was used. Within each cycle, a full-scan MS spectra were acquired firstly in the Orbitrap at a mass resolving power of 120,000 (at m/z 200) across an m/z range of 300–2000 using quadrupole isolation, an automatic gain control (AGC) target of 4e5 and a maximum injection time of 50 milliseconds, followed by higher-energy collisional dissociation (HCD)-MS/MS at a mass resolving power of 15,000 (at m/z 200), a normalised collision energy (NCE) of 27% at positive mode and 30% at negative mode, an m/z isolation window of 1, a maximum injection time of 22 milliseconds and an AGC target of 5e4. For the improved structural characterisation of glycerophosphocholine (PC) lipid cations, a data-dependent product ion (m/z 184.0733)-triggered collision-induced dissociation (CID)-MS/MS scan was performed in the cycle using a q-value of 0.25 and a NCE of 30%, with other settings being the same as that for HCD-MS/MS. For the improved structural characterisation of triacylglycerol (TG) lipid cations, the fatty acid + NH3 neutral loss product ions observed by HCD-MS/MS were used to trigger the acquisition of the top-3 data-dependent CID-MS3 scans in the cycle using a q-value of 0.25 and a NCE of 30%, with other settings being the same as that for HCD-MS/MS. Identification and quantification of lipids LC-MS/MS data was searched through MS Dial 4.48. The mass accuracy settings are 0.005 Da and 0.025 Da for MS1 and MS2. The minimum peak height is 50000 and mass slice width is 0.05 Da. The identification score cut off is 80%. Post identification was done with a text file containing name and m/z of each standard in SPLASH® II LIPIDOMIX® Mass Spec Standard. In positive mode, [M+H]+, [M+NH4]+ and [M+H-H2O]+ were selected as ion forms and lipid classes including CAR, LPC, LPE, PC, PE, PS, CL, EtherLPC, EtherLPE, EtherPC, EtherPE, Sph, DHSph, SM, MG, DG, EtherDG, TG, EtherTG, CE, CoQ, Cer_NS, Cer_NDS, CerP, HexCer_NS, HexCer_NDS, Hex2Cer, Hex3Cer and ST were selected for the search. In negative mode, [M-H]- and [M+CH3COO]- were selected as ion forms and lipid classes including LPS, LPG, LPI, LPA, PA, PC, PE, PG, PI, PS, CL, EtherPC, EtherPE, EtherPS, EtherPI, EtherPG, EtherLPG, SM, Cer_NS, Cer_NDS, CerP, HexCer_NS, HexCer_NDS, Hex2Cer, Hex3Cer, SHexCer, NAE and GM3 were selected for the search. The retention time tolerance for alignment is 0.1 min. The peak count filter is 50% and the N% detected in at least one group is 66%. Lipids with maximum intensity less than 5-fold of average intensity in blank was removed. All other settings were default. All lipid LC-MS features were manually inspected and re-integrated when needed. These four types of lipids, 1) Cer_NS, Cer_NDS and CL with only sum composition, 2) lipid identification due to peak tailing, 3) retention time outliner within each lipid class, 4) LPA and PA generated by in-source fragmentation of LPS and PS were also removed. Relative quantification of lipid species was achieved by comparison of the LC peak areas of identified lipids against those of the corresponding internal lipid standards in the same lipid class, and the resultant ratio of peak area was then normalized to weight of tissue and total PC content. For the lipid classes without correspondent isotope-labelled internal lipid standards, the LC peak areas of individual molecular species within these classes were normalised as follows: the MG species against the DG (18:1D7_15:0) internal standard; the CL, LPG, PG, LPA and PA against the PI (18:1D7_15:0) internal standard; the LPS against the PS (18:1D7_15:0) internal standard; the Hex1Cer against the SM (d36:2D9) internal standard. Given that only a single lipid standard per class was used, some of the identified lipids were normalised against a standard from a different class or sub-class, and no attempts were made to quantitatively correct for different ESI responses of individual lipids due to concentration, acyl chain length, degree of unsaturation, or matrix effects caused by differences in chromatographic retention times compared with the relevant standards. The results reported here are for relative quantification and should not be considered to reflect the absolute concentrations of each lipid or lipid sub-class. Lipidomics data analysis All lipidomics data were further processed with Perseus (Version 1.5.0.40). Within Perseus, values were log2 transformed, each lipid species normalized to the z-score, and the replicates grouped accordingly. All lipid species that had less than 70 percent of “valid value” in each group were removed and the missing values were replaced by imputation. A two-sample t-test (FDR < 5%) was performed to obtain a list of significantly regulated lipids between diets within each strain. Lastly, lipidomics data were analysed for biophysical data, lipid functions and organelle associations using the Lipid Ontology (LION) enrichment analysis web application, as well as BioPAN (Bioinformatics Methodology For Pathway Analysis).
Ion Mode:POSITIVE
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