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MB Sample ID: SA343058
| Local Sample ID: | young_6 |
| Subject ID: | SU003287 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | Young donors (aged 25 years or younger) and older donors (aged 50 years or older). |
| Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003287 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | Young donors (aged 25 years or younger) and older donors (aged 50 years or older). |
| Gender: | Male and female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| young_6 | SA343058 | FL040527 | Heart | Sample source |
| young_6 | SA343058 | FL040527 | M | Sex |
| young_6 | SA343058 | FL040527 | young | Age group |
Collection:
| Collection ID: | CO003280 |
| Collection Summary: | Control donor hearts that were considered suitable for transplantation but ultimately not used due to factors such as transport issues, immune mismatches, and size discrepancies between donor and recipient, were collected in this study. These hearts came from individuals who died of non-cardiac reasons and had no risk factors for heart disease, including having a BMI under 30. Following a thorough pathological review, these hearts were confirmed to be structurally normal through histological evaluation. Left ventricular (LV) samples were taken directly in the operating room, immediately snap-frozen in liquid nitrogen at -196°C, and then stored at the Sydney Heart Bank at the University of Sydney, at temperatures ranging from -170 to -180°C. This collection process received ethical approval from the University of Sydney's Ethics Committee (USYD # 2021/122). |
| Collection Protocol ID: | CO003212 |
| Sample Type: | Heart |
| Storage Conditions: | Described in summary |
Treatment:
| Treatment ID: | TR003296 |
| Treatment Summary: | The samples detailed in this study did not undergo any treatment. |
| Treatment Protocol ID: | TR003228 |
Sample Preparation:
| Sampleprep ID: | SP003294 |
| Sampleprep Summary: | Lipids were extracted from ~20 mg heart tissue using a two-phase method with methyl-tert-butyl ether (MTBE) and water. Tissue was homogenised with steel beads in 250 µL methanol containing 0.01% (w/v) butylhydroxytoluene (BHT) and mass spectrometry internal standards: 2 nmoles PC(19:0/19:0); 1 nmole each of SM(d18:1/12:0), GluCer(d18:1/12:0), Cer(d18:1/17:0), PS(17:0/17:0), PE(17:0/17:0), PA(17:0/17:0), PI(d7-18:1/15:0), PG(17:0/17:0), CL(14:0/14:0/14:0/14:0), and TG(17:0/17:0/17:0); 0.5 nmoles each of DG(d7-18:1/15:0), CholE(17:0), LPC (17:0), LPE(17:1), and AcCa(d3-16:0); and 0.2 nmole each of Sph(d17:1), S1P(d17:1), LacCer(d18:1/12:0), and MG(d7-18:1). MTBE (850 µL) was added, and samples were sonicated in a 4°C water bath for 30 min. Mass spectrometry grade water (212 µL) was added to induce phase separation after vortexing and centrifugation at 2000g for 5 min. The upper organic phase was collected in 5 mL glass tubes and the aqueous phase was extracted twice more with 500 µL MTBE and 150 µL methanol followed by sonication for 15 min and phase separation with 125 µL water. Organic phases from the three extractions were combined and dried under vacuum in a Savant SC210 SpeedVac (ThermoFisher Scientific). Lipids were reconstituted in 400 µL 80% methanol/20% water/0.1% formic acid containing 0.01% (w/v) BHT and stored at -80 °C. |
| Sampleprep Protocol ID: | SP003225 |
Combined analysis:
| Analysis ID | AN005198 | AN005199 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Waters HPLC C18 (100 x 2.1mm, 1.7um) | Waters HPLC C18 (100 x 2.1mm, 1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive HF-X Orbitrap | Thermo Q Exactive HF-X Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | pmoles/mg tissue | nanomoles/mg of tissue |
Chromatography:
| Chromatography ID: | CH003933 |
| Chromatography Summary: | A ThermoFisher Q-Exactive HF-X mass spectrometer coupled to a Vanquish HPLC with a Waters Acquity UPLC BEH C18 (100 x 2.1 mm, 1.7 um) column was used for LC-MS/MS, with minor modifications to our previously-reported method. HPLC solvent A was 10 mM ammonium formate, 0.1% formic acid in acetonitrile:water (60:40), and solvent B was 10 mM ammonium formate, 0.1% formic acid in isopropanol:acetonitrile (90:10). A 27 min binary gradient at 0.28 mL/min was used: 0 min, 80:20 A/B; 3 min, 80:20 A/B; 5.5 min, 55:45 A/B; 8 min, 35:65 A/B; 13 min, 15:85 A/B; 14 min, 0:100 A/B; 20 min, 0:100 A/B; 20.2 min, 70:30 A/B; 27 min, 70:30 A/B. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters HPLC C18 (100 x 2.1mm, 1.7um) |
| Column Temperature: | 32 |
| Flow Gradient: | 0 min, 80:20 A/B; 3 min, 80:20 A/B; 5.5 min, 55:45 A/B; 8 min, 35:65 A/B; 13 min, 15:85 A/B; 14 min, 0:100 A/B; 20 min, 0:100 A/B; 20.2 min, 70:30 A/B; 27 min, 70:30 A/B |
| Flow Rate: | 0.28 mL/min |
| Solvent A: | 60% acetonitrile/40% water; 10 mM ammonium formate; 0.1% formic acid |
| Solvent B: | 90% isopropanol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004931 |
| Analysis ID: | AN005198 |
| Instrument Name: | Thermo Q Exactive HF-X Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Data was acquired in full scan/data-dependent MS2 mode (full scan resolution 60,000 FWHM, scan range 220–1600 m/z). Sample order was randomised, and data was collected in both positive and negative mode for each sample. The ten most abundant ions in each cycle were subjected to MS2, with an isolation window of 1.4 m/z, collision energy 30 eV, resolution 17,500 FWHM, maximum integration time 110 ms and dynamic exclusion window 10 s. An exclusion list of background ions was used based on a solvent blank. An inclusion list was used for all internal standards. LipidSearch software (version 4.2, Thermo Fisher) was used for lipid annotation, chromatogram alignment, and peak integration. Lipid annotation required both accurate precursor ion mass (tolerance 5 ppm) and diagnostic product ions (tolerance 8 ppm). Individual lipids were expressed as ratios to the class-specific internal standard, then multiplied by the amount of internal standard to calculate molar amounts for each lipid. Lipid levels were expressed as nmoles/mg tissue. |
| Ion Mode: | POSITIVE |
| MS ID: | MS004932 |
| Analysis ID: | AN005199 |
| Instrument Name: | Thermo Q Exactive HF-X Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Data was acquired in full scan/data-dependent MS2 mode (full scan resolution 60,000 FWHM, scan range 220–1600 m/z). Sample order was randomised, and data was collected in both positive and negative mode for each sample. The ten most abundant ions in each cycle were subjected to MS2, with an isolation window of 1.4 m/z, collision energy 30 eV, resolution 17,500 FWHM, maximum integration time 110 ms and dynamic exclusion window 10 s. An exclusion list of background ions was used based on a solvent blank. An inclusion list was used for all internal standards. LipidSearch software (version 4.2, Thermo Fisher) was used for lipid annotation, chromatogram alignment, and peak integration. Lipid annotation required both accurate precursor ion mass (tolerance 5 ppm) and diagnostic product ions (tolerance 8 ppm). Individual lipids were expressed as ratios to the class-specific internal standard, then multiplied by the amount of internal standard to calculate molar amounts for each lipid. Lipid levels were expressed as nmoles/mg tissue. |
| Ion Mode: | NEGATIVE |
