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MB Sample ID: SA242384
| Local Sample ID: | 20220508_pos_KO_rep3_16ul |
| Subject ID: | SU002511 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Strain Details: | HEK293T |
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Subject:
| Subject ID: | SU002511 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Strain Details: | HEK293T |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 20220508_pos_KO_rep3_16ul | SA242384 | FL030359 | UBXD8 Knockout | Description |
| 20220508_pos_KO_rep3_16ul | SA242384 | FL030359 | 16 | inj vol |
Collection:
| Collection ID: | CO002504 |
| Collection Summary: | Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and streptomycin. Cells were maintained in a humidified, 5 % CO2 atmosphere at 37°C. The CRISPR-Cas9 gene editing system was used to generate UBXD8 knockout cell lines in HEK293T cells. Cells were seeded into four 150 mm TC dishes. Cells were lysed in Homogenization buffer (225 mM mannitol, 75 mM sucrose, and 30 mM Tris-Cl, pH 7.4) using a Dounce homogenizer. The lysate was centrifuged three times at 600xg for 5 minutes to remove unlysed cells and nuclei resulting in post-nuclear supernatants (PNS). The cleared lysate was centrifuged at 7000xg to separate crude mitochondrial pellet and supernatant containing microsomes. The supernatant was cleared by centrifugation at 20,000xg for 30 minutes followed by microsome isolation using high-speed centrifugation at 100,000xg for 1 hour. The crude mitochondrial pellet was washed twice in homogenization buffer containing 0.1 mM EGTA at 7000xg and 10,000xg for 10 minutes. MAMs were isolated from crude mitochondria using 30 % Percoll gradient centrifugation at 95,000xg for 1 hr in a swinging-bucket rotor. The banded MAM fraction was washed once with phosphate-buffered saline (PBS) before lysing in lysis buffer (50 mM Tris-Cl, pH 7.2, 150 mM NaCl). The pure mitochondrial fractions were resuspended and washed in mitochondrial resuspension buffer (250 mM mannitol, 0.5 mM EGTA, 5 mM HEPES pH7.4). Protein concentrations for both soluble and pellet fractions were determined by DC protein assay kit (Biorad). For lipidomics of MAMs, the final banded MAM fraction was washed twice with liquid chromatography-mass spectrometry (LC-MS) grade PBS and the final MAM pellet was resuspended in LC-MS grade PBS. Next, 1mL of cold 50% methanol was added and the MAMS were transferred to glass vials. Chloroform was added (0.5mL) and the mixture was gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. Lipids were transferred to a clean glass vial using a glass Hamilton syringe. Lipids were extracted twice using chloroform prior to being dried under nitrogen gas. Samples were normalized according to protein concentration. Extracted lipids were resuspended in a 1:1:1 solution of methanol:chloroform:isopropanol prior to mass spectrometry (MS) analysis. |
| Sample Type: | Epithelial cells |
Treatment:
| Treatment ID: | TR002523 |
| Treatment Summary: | Wild-type vs UBXD8 Knockout |
Sample Preparation:
| Sampleprep ID: | SP002517 |
| Sampleprep Summary: | Lipids were isolated from collected cultured cells. Cells were washed with PBS, treated with cold 50% methanol (1mL) and transferred to glass vials. Next, chloroform (0.5mL) was added and samples were gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. Lipids were transferred to a clean glass vial using a glass Hamilton syringe. Lipids were extracted twice using chloroform prior to being dried under nitrogen gas. Samples were normalized according to protein concentration when resuspended in a 1:1:1 solution of methanol:chloroform:isopropanol prior to mass spectrometry (MS) analysis. To assist quantification various volumes were injected for MS (4, 8, and 16ul for positive mode analysis and 5, 10, and 15ul for negative mode analysis). |
Combined analysis:
| Analysis ID | AN003944 | AN003945 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um) | Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH002920 |
| Chromatography Summary: | Lipids were identified and quantitatively measured using ultra high-performance liquid-chromatography high-resolution tandem MS/MS (UHPLC-MS/MS) as recently described89,90. Separation of lipids was done by reverse-phase chromatography using a Waters ACQUITY CSH C18 column (150x2.1mm; 1.7um) at 60°C using a Vanquish UHPLC system (Thermo Scientific) and two solvents: solvent A (40:60 water-methanol plus 10mM ammonium formate and 0.1% formic acid) and solvent B (10:90 methanol-isopropanol plus 10mM ammonium formate and 0.1% formic acid). UHPLC was performed at a 0.25 ml per min flow rate for 30 min per sample, starting at 25% solvent B and ending at 100% solvent B as described. The column was washed and equilibrated between samples. Samples were run in a semi-random order where WT or UBXD8 KO samples were interspersed with blank samples. Each sample was repeatedly analyzed using various injection volumes (4, 8, 16ul for positive mode and 5, 10, and 15ul for negative mode). |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 60 |
| Flow Gradient: | 25% to 100% |
| Flow Rate: | 0.25mL per min |
| Solvent A: | 40% water; 60% methanol 10mM ammonium formate and 0.1% formic acid |
| Solvent B: | 10% methanol; 90% isopropanol 10mM ammonium formate and 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003680 |
| Analysis ID: | AN003944 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Samples were run in a semi-random order where WT or UBXD8 KO samples were interspersed with blank samples. Lipids were ionized using a heated electrospray ionization (HESI) source and nitrogen gas and measured using a Q-Exactive Plus mass spectrometer operating at a MS1 resolution of either 70,000 or 140,000 and a MS2 resolution of 35,000. MS1 Spectra were collected over a mass range of 200 to 1,600 m/z with an automatic gain control (AGC) setting of 1e6 and transient times of 250 ms (70,000 resolution) or 520 ms (140,000 resolution). MS2 spectra were collected using a transient time of 120 ms and an AGC setting of 1e5. Each sample was analyzed using negative and positive ion modes. The mass analyzer was calibrated weekly. SPLASH LIPIDOMIX mass spectrometry standards (Avanti Polar Lipids) were used in determining extraction efficiencies and lipid quantitation. Lipids were identified and quantified using MAVEN, and El-MAVEN (Elucidata). UHPLC retention time, MS1 peaks, and MS2 fragments were used to identify lipids. Lipids were included if they were observed in 3-6 samples in both UBXD8 KO and WT cells. Missing values in a sample were not imputed. The following lipid classes were included in the analysis: cholesteryl esters (CE), diacylglycerol (DG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and triacylglycerol (TG). Guidelines from the Lipidomic Standards Initiative were followed for lipid species identification and quantification, including consideration of isotopic patterns resulting from naturally occurring 13C atoms and isomeric overlap. The following MS2 information was used to confirm each lipid species: PC fragment of 184.073 (positive mode) and tail identification using formic adduct (negative mode); PE fragment of 196.038 or the tail plus 197.046 (negative mode) and neutral loss (NL) of 141.019 (positive mode); PG fragment of 152.996 plus the identification of the FA tails (negative mode) and NL 189.04 of [M+NH4]+ adduct (positive mode); PI fragment of 241.012 (negative) and NL 277.056 of [M+NH4]+ adduct (positive mode); PS NL of 87.032 (negative); DG and TG by NL of FA tails (positive mode); and CE fragment of 369.352 or neutral loss of 368.35 (positive). |
| Ion Mode: | POSITIVE |
| MS ID: | MS003681 |
| Analysis ID: | AN003945 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Samples were run in a semi-random order where WT or UBXD8 KO samples were interspersed with blank samples. Lipids were ionized using a heated electrospray ionization (HESI) source and nitrogen gas and measured using a Q-Exactive Plus mass spectrometer operating at a MS1 resolution of either 70,000 or 140,000 and a MS2 resolution of 35,000. MS1 Spectra were collected over a mass range of 200 to 1,600 m/z with an automatic gain control (AGC) setting of 1e6 and transient times of 250 ms (70,000 resolution) or 520 ms (140,000 resolution). MS2 spectra were collected using a transient time of 120 ms and an AGC setting of 1e5. Each sample was analyzed using negative and positive ion modes. The mass analyzer was calibrated weekly. SPLASH LIPIDOMIX mass spectrometry standards (Avanti Polar Lipids) were used in determining extraction efficiencies and lipid quantitation. Lipids were identified and quantified using MAVEN, and El-MAVEN (Elucidata). UHPLC retention time, MS1 peaks, and MS2 fragments were used to identify lipids. Lipids were included if they were observed in 3-6 samples in both UBXD8 KO and WT cells. Missing values in a sample were not imputed. The following lipid classes were included in the analysis: cholesteryl esters (CE), diacylglycerol (DG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and triacylglycerol (TG). Guidelines from the Lipidomic Standards Initiative were followed for lipid species identification and quantification, including consideration of isotopic patterns resulting from naturally occurring 13C atoms and isomeric overlap. The following MS2 information was used to confirm each lipid species: PC fragment of 184.073 (positive mode) and tail identification using formic adduct (negative mode); PE fragment of 196.038 or the tail plus 197.046 (negative mode) and neutral loss (NL) of 141.019 (positive mode); PG fragment of 152.996 plus the identification of the FA tails (negative mode) and NL 189.04 of [M+NH4]+ adduct (positive mode); PI fragment of 241.012 (negative) and NL 277.056 of [M+NH4]+ adduct (positive mode); PS NL of 87.032 (negative); DG and TG by NL of FA tails (positive mode); and CE fragment of 369.352 or neutral loss of 368.35 (positive). |
| Ion Mode: | NEGATIVE |
