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MB Sample ID: SA246006
| Local Sample ID: | 531_n_SFG_GM |
| Subject ID: | SU002546 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002546 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 531_n_SFG_GM | SA246006 | FL030870 | GRN | Genotype |
| 531_n_SFG_GM | SA246006 | FL030870 | SFG_GM | Brain region |
Collection:
| Collection ID: | CO002539 |
| Collection Summary: | Frozen brain tissue was homogenised by bead beating at 4°C in ice cold HEPES buffer (50mM, pH 7.4) plus 5 mM NaF, 2 mM Na3VO4, 10 mM KCl and cOmplete Mini EDTA-free Protease Inhibitor Cocktail |
| Sample Type: | Brain |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002558 |
| Treatment Summary: | Samples were obtained from frontotemporal dementia cases with inherited gene mutations in either GRN, or C9orf72 gene repeat expansion carriers and controls. |
Sample Preparation:
| Sampleprep ID: | SP002552 |
| Sampleprep Summary: | Lipids were extracted from 100uL of brain homogenate using a two-phase methyl-tert-butyl ether (MTBE)/methanol/water protocol as described by Matyash et al. (2008), with the addition of the following internal standards: 2000 pmoles SM(d18:1_12:0), 2000 pmoles GluCer(d18:1_12:0), 500 pmoles LacCer(d18:1_12:0), 500 pmoles ST(18:1_17:0), 500 pmoles Cer(18:1/17:0), 200 pmoles C17:0 Sph, 200 pmoles C17:1 S1P, 200 pmoles d3-C16 AcCa, 500 pmoles C17:1 LPE, 500 pmoles C17:1 LPS, 200 pmoles C17:0 LPA, 500 pmoles C17:0 LPC, 5000 pmoles PC(19:0_19:0), 2000 pmoles PS(17:0_17:0), 2000 pmoles C17:0 PE, 2000 pmoles C17:0 PG, 1000 pmoles C17:0 PA, 1000 pmoles d7-18:1_15:0 PI, 2000 pmoles CL(14:0_14:0_14:0_14:0), 2000 pmoles TAG(17:0_17:0_17:0), 500 pmoles DAG d7- 18:1/15:0), 500 pmoles d7- 18:1 MAG, 2000 pmoles C17:0 CholE, 1000 pmoles d7 Chol. Lipids were reconstituted in 400uL of 100% HPLC methanol, then diluted 1/5 in 80% v/v HPLC methanol, containing 1 mM ammonium formate and 0.2% formic acid. |
Combined analysis:
| Analysis ID | AN004007 | AN004008 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC CSH 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 protein | pmoles/mg protein |
Chromatography:
| Chromatography ID: | CH002959 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0 min, 80:20 A/B; 3 min, 80:20 A/B; 5.5 min, 55:45 A/B; 8 min, 36:65 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; 0.1% formic acid; 10 mM ammonium formate |
| Solvent B: | 90% isopropanol/ 10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003754 |
| Analysis ID: | AN004007 |
| Instrument Name: | Thermo Q Exactive HF-X Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS data was acquired in full scan/data-dependent MS2 (full scan resolution 60,000 FWHM, scan range 220–1600 m/z) in both positive and negative ionization modes. 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 of the [M+H]+ and [M-H]- ions was used for all internal standards. LipidSearch software v4.2 (Thermo Fisher) was used for lipid annotation, chromatogram alignment, and peak integration from extracted ion chromatograms. Lipid annotation was based on precursor and product ions in both positive and negative ion mode. Individual lipids were expressed as ratios to an internal standard specific for each lipid class, then multiplied by the amount of internal standard added to produce a molar amount of each lipid per sample, which was normalised to protein amount in each sample. |
| Ion Mode: | POSITIVE |
| MS ID: | MS003755 |
| Analysis ID: | AN004008 |
| Instrument Name: | Thermo Q Exactive HF-X Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS data was acquired in full scan/data-dependent MS2 (full scan resolution 60,000 FWHM, scan range 220–1600 m/z) in both positive and negative ionization modes. 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 of the [M+H]+ and [M-H]- ions was used for all internal standards. LipidSearch software v4.2 (Thermo Fisher) was used for lipid annotation, chromatogram alignment, and peak integration from extracted ion chromatograms. Lipid annotation was based on precursor and product ions in both positive and negative ion mode. Individual lipids were expressed as ratios to an internal standard specific for each lipid class, then multiplied by the amount of internal standard added to produce a molar amount of each lipid per sample, which was normalised to protein amount in each sample. |
| Ion Mode: | NEGATIVE |
