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.
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 | ST003326 |
| Study Title | Lipidome profiling in non-alcoholic steatohepatitis identifies phosphatidylserine synthase 1 as a regulator of hepatic lipoprotein metabolism |
| Study Summary | Non-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 Name | Montgomery |
| First Name | Magdalene |
| Address | Corner Grattan Street & Royal Parade |
| magdalene.montgomery@unimelb.edu.au | |
| Phone | 0422059907 |
| Submit Date | 2024-05-13 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | abf |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-10-21 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
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 |
