Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA161885

Local Sample ID:	Mito_002_ne
Subject ID:	SU001794
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Genotype Strain:	HeLa cell line
Gender:	Not applicable

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Subject:

Subject ID:	SU001794
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Genotype Strain:	HeLa cell line
Gender:	Not applicable

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Mito_002_ne	SA161885	FL019126	Mock(+)	Group

Collection:

Collection ID:	CO001787
Collection Summary:	Mitochondria were isolated from HeLa cells using a mitochondria isolation kit for tissues (cat. no. 89874, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Mitochondrial pellets were washed and stored in 1xTBS buffer supplemented with 0.1% CHAPS on ice until further use. Isolated mitochondrial fractions were used for lipidomic analyses.
Sample Type:	HeLa cells

Treatment:

Treatment ID:	TR001807
Treatment Summary:	Conditional knockdown and reconstitution of PTPIP51 in HeLa cells were performed as previously reported (Bong et al, 2020). Huma PTPIP51-targeting small hairpin RNA (shRNA) sequences (5’-ATGACTTGATGCCACTATTTA-3’) were inserted into the Tet-pLKO-blasticidin vector. Lentiviruses were produced according to a method described previously (Kim et al, 2018). HeLa cells infected with lentiviruses were selected with blasticidin (10 μg/ml, Invitrogen, USA) for at least 7 days and named HeLa Tet-on-shPTPIP51 cells. The genes encoding human full-length PTPIP51 and PTPIP51_ΔFFAT were cloned into the pCAG-Flag-IRES-puro vector. HeLa Tet-on-shPTPIP51 cells were transfected with pCAG-Flag-IRES-puro empty vector (Mock), PTPIP51, and PTPIP51_ΔFFAT using Lipofectamine 3000 (Life Technologies, USA). Transfected cells were selected with puromycin (2 μg/ml, Amresco, USA) for at least 4 days. For endogenous PTPIP51 knockdown, doxycycline (1 μg/ml, Sigma-Aldrich, USA) was added every two days. Because the shRNAs were designed to target the 3’ UTR of PTPIP51, exogenously added constructs were not targeted.

Treatment ID:

TR001807

Treatment Summary:

Conditional knockdown and reconstitution of PTPIP51 in HeLa cells were performed as previously reported (Bong et al, 2020). Huma PTPIP51-targeting small hairpin RNA (shRNA) sequences (5’-ATGACTTGATGCCACTATTTA-3’) were inserted into the Tet-pLKO-blasticidin vector. Lentiviruses were produced according to a method described previously (Kim et al, 2018). HeLa cells infected with lentiviruses were selected with blasticidin (10 μg/ml, Invitrogen, USA) for at least 7 days and named HeLa Tet-on-shPTPIP51 cells. The genes encoding human full-length PTPIP51 and PTPIP51_ΔFFAT were cloned into the pCAG-Flag-IRES-puro vector. HeLa Tet-on-shPTPIP51 cells were transfected with pCAG-Flag-IRES-puro empty vector (Mock), PTPIP51, and PTPIP51_ΔFFAT using Lipofectamine 3000 (Life Technologies, USA). Transfected cells were selected with puromycin (2 μg/ml, Amresco, USA) for at least 4 days. For endogenous PTPIP51 knockdown, doxycycline (1 μg/ml, Sigma-Aldrich, USA) was added every two days. Because the shRNAs were designed to target the 3’ UTR of PTPIP51, exogenously added constructs were not targeted.

Sample Preparation:

Sampleprep ID:	SP001800
Sampleprep Summary:	For mitochondrial analysis, isolated mitochondria from 2x10^7 cells were mixed with 500 µl of 100 mM hydrochloric acid solution in methanol/water (8:2, v/v), 700 µl of chloroform and 500 µl of 100 mM hydrochloric acid solution in water. The mitochondrial sample was homogenized with 2.8 mm zirconium oxide beads for 5 min. After centrifugation at 30,130 xg and 4°C for 15 min, 600 µl of the lower phase was collected. Both the cell and mitochondrial extracts were dried under a gentle nitrogen stream at room temperature and reconstituted in 300 µL of isopropanol/acetonitrile/water (2:1:1, v/v/v). Eighty microliters of each sample was mixed with 20 µL of SPLASH LIPIDOMIX internal standard mix (Avanti Polar Lipids, USA). Finally, 5 µl of each sample was injected into the UPLC-QTOF MS system.

Sampleprep ID:

SP001800

Sampleprep Summary:

For mitochondrial analysis, isolated mitochondria from 2x10^7 cells were mixed with 500 µl of 100 mM hydrochloric acid solution in methanol/water (8:2, v/v), 700 µl of chloroform and 500 µl of 100 mM hydrochloric acid solution in water. The mitochondrial sample was homogenized with 2.8 mm zirconium oxide beads for 5 min. After centrifugation at 30,130 xg and 4°C for 15 min, 600 µl of the lower phase was collected. Both the cell and mitochondrial extracts were dried under a gentle nitrogen stream at room temperature and reconstituted in 300 µL of isopropanol/acetonitrile/water (2:1:1, v/v/v). Eighty microliters of each sample was mixed with 20 µL of SPLASH LIPIDOMIX internal standard mix (Avanti Polar Lipids, USA). Finally, 5 µl of each sample was injected into the UPLC-QTOF MS system.

Combined analysis:

Analysis ID	AN002797	AN002798
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Waters Acquity I-Class	Waters Acquity I-Class
Column	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	ABI Sciex 5600 TripleTOF	ABI Sciex 5600 TripleTOF
Ion Mode	POSITIVE	NEGATIVE
Units	Peak area	Peak area

Chromatography:

Chromatography ID:	CH002067
Chromatography Summary:	Chromatographic separation of lipids in cells and mitochondria was performed with an Acquity UPLC system (Waters, USA) using an Acquity UPLC CSH C18 column (2.1100 mm, 1.7 µm; Waters) at 55°C and a flow rate of 0.4 ml/min. The mobile phase for positive ion mode comprised 10 mM ammonium formate in water/acetonitrile (40:60, v/v) containing 0.1% formic acid (solvent A) and isopropanol/acetonitrile (90:10, v/v) containing 0.1% formic acid (solvent B). The mobile phase for negative ion mode comprised 10 mM ammonium acetate in water/acetonitrile (60:40, v/v) (solvent A) and isopropanol/acetonitrile (90:10, v/v) (solvent B). The UPLC gradient was programmed as follows: 40% to 43% B from 0 min to 2 min, 43% to 50% B from 2 min to 2.1 min, 50% to 54% B from 2.1 min to 12 min, 54% to 70% B from 12 min to 12.1 min, 70% to 99% B from 12.1 min to 18 min, 99% to 40% B from 18 min to 18.1 min, and 40% B for 2 min to equilibrate for the next run.
Instrument Name:	Waters Acquity I-Class
Column Name:	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:	55
Flow Gradient:	40% to 43% B from 0 min to 2 min, 43% to 50% B from 2 min to 2.1 min, 50% to 54% B from 2.1 min to 12 min, 54% to 70% B from 12 min to 12.1 min, 70% to 99% B from 12.1 min to 18 min, 99% to 40% B from 18 min to 18.1 min, and 40% B for 2 min to equilibrate for the next run.
Flow Rate:	0.4 ml/min
Solvent A:	40% water/60acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Solvent B:	90% isopropanol/10% acetonitrile
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002592
Analysis ID:	AN002797
Instrument Name:	ABI Sciex 5600 TripleTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	m/z range of 80-1500 The following parameter settings were used: ion spray voltage, 5500 V (positive mode) and 4500 V (negative mode); source temperature, 500°C; nebulizer gas pressure, 50 psi; drying gas pressure, 60 psi; and curtain gas pressure, 30 psi. An atmospheric pressure chemical ionization calibration solvent was used to maintain mass accuracy with an automated calibrant delivery system (Sciex). Information-dependent acquisition (IDA) was used to acquire MS/MS spectra for ions. All samples were pooled in equal amounts to generate quality control (QC) samples, which were injected after every 4 samples to calculate the coefficient of variation (CV) and assess analytical reproducibility.
Ion Mode:	POSITIVE

MS ID:	MS002593
Analysis ID:	AN002798
Instrument Name:	ABI Sciex 5600 TripleTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	m/z range of 80-1500 The following parameter settings were used: ion spray voltage, 5500 V (positive mode) and 4500 V (negative mode); source temperature, 500°C; nebulizer gas pressure, 50 psi; drying gas pressure, 60 psi; and curtain gas pressure, 30 psi. An atmospheric pressure chemical ionization calibration solvent was used to maintain mass accuracy with an automated calibrant delivery system (Sciex). Information-dependent acquisition (IDA) was used to acquire MS/MS spectra for ions. All samples were pooled in equal amounts to generate quality control (QC) samples, which were injected after every 4 samples to calculate the coefficient of variation (CV) and assess analytical reproducibility.
Ion Mode:	NEGATIVE