Summary of Study ST001717

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 PR001101. The data can be accessed directly via it's Project DOI: 10.21228/M8CT3Q 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.

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Study ID	ST001717
Study Title	Phospholipid transfer function of PTPIP51 at mitochondria-associated ER membranes
Study Summary	In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determined the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB protein at MAMs. The structure of PTPIP51_TPR showed an archetypal TPR fold, and an electron density corresponding to an unidentified lipid-like molecule probably derived from the protein expression host was found in the structure. We revealed functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduced the mitochondrial cardiolipin level. Additionally, we confirmed that the PTPIP51–VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM-tethering function similar to the ERMES complex in yeast.
Institute	Korea Basic Science Institute
Department	Western Seoul Center
Laboratory	Integrated Metabolomics Research Group
Last Name	Lee
First Name	Jueun
Address	150, Bugahyeon-ro, Seodaemun-gu, Seoul, Republic of Korea (Zip code: 03759)
Email	lje3080@kbsi.re.kr
Phone	+82-2-6908-6256
Submit Date	2021-03-04
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2021-03-16
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001101
Project DOI:	doi: 10.21228/M8CT3Q
Project Title:	Phospholipid transfer function of PTPIP51 at mitochondria-associated ER membranes
Project Type:	MS-based lipid profiling
Project Summary:	LC/MS-based lipid profiling of mitochondria obtained HeLa cell line
Institute:	Korea Basic Science Institute
Department:	Western Seoul Center
Laboratory:	Integrated Metabolomics Research Group
Last Name:	Lee
First Name:	Jueun
Address:	150, Bugahyeon-ro, Seoul, Seoul, 03759, Korea, South
Email:	lje3080@kbsi.re.kr
Phone:	+82-2-6908-6256

Subject:

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

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Group
SA161865	Mito_008_po	FFAT(+)
SA161866	Mito_012_po	FFAT(+)
SA161867	Mito_012_ne	FFAT(+)
SA161868	Mito_004_ne	FFAT(+)
SA161869	Mito_004_po	FFAT(+)
SA161870	Mito_008_ne	FFAT(+)
SA161871	Mito_003_ne	FL(+)
SA161872	Mito_010_po	FL(+)
SA161873	Mito_007_ne	FL(+)
SA161874	Mito_007_po	FL(+)
SA161875	Mito_003_po	FL(+)
SA161876	Mito_010_ne	FL(+)
SA161877	Mito_005_ne	Mock
SA161878	Mito_009_ne	Mock
SA161879	Mito_001_po	Mock
SA161880	Mito_001_ne	Mock
SA161881	Mito_005_po	Mock
SA161882	Mito_009_po	Mock
SA161883	Mito_006_po	Mock(+)
SA161884	Mito_002_po	Mock(+)
SA161885	Mito_002_ne	Mock(+)
SA161886	Mito_011_po	Mock(+)
SA161887	Mito_006_ne	Mock(+)
SA161888	Mito_011_ne	Mock(+)
SA161889	tQC_mito_03_ne	QC
SA161890	QC_mito_03_ne	QC
SA161891	QC_mito_02_po	QC
SA161892	QC_mito_02_ne	QC
SA161893	tQC_mito_03_po	QC
SA161894	QC_mito_03_po	QC
SA161895	QC_mito_01_po	QC
SA161896	QC_mito_01_ne	QC

Showing results 1 to 32 of 32

Showing results 1 to 32 of 32

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	Triple TOF	Triple TOF
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:	Triple TOF
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:	Triple TOF
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