{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001717","ANALYSIS_ID":"AN002797","VERSION":"1","CREATED_ON":"March 4, 2021, 6:40 am"},

"PROJECT":{"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"},

"STUDY":{"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"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","GENDER":"Not applicable"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Mito_001_po",
"Factors":{"Group":"Mock"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_001_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_002_po",
"Factors":{"Group":"Mock(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_002_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_003_po",
"Factors":{"Group":"FL(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_003_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_004_po",
"Factors":{"Group":"FFAT(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_004_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_005_po",
"Factors":{"Group":"Mock"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_005_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_006_po",
"Factors":{"Group":"Mock(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_006_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_011_po",
"Factors":{"Group":"Mock(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_011_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_007_po",
"Factors":{"Group":"FL(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_007_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_008_po",
"Factors":{"Group":"FFAT(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_008_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_012_po",
"Factors":{"Group":"FFAT(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_012_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_009_po",
"Factors":{"Group":"Mock"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_009_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_010_po",
"Factors":{"Group":"FL(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_010_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"QC_mito_01_po",
"Factors":{"Group":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_mito_01_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"QC_mito_02_po",
"Factors":{"Group":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_mito_02_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"QC_mito_03_po",
"Factors":{"Group":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_mito_03_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"tQC_mito_03_po",
"Factors":{"Group":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"tQC_mito_03_po.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_001_ne",
"Factors":{"Group":"Mock"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_001_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_002_ne",
"Factors":{"Group":"Mock(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_002_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_003_ne",
"Factors":{"Group":"FL(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_003_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_004_ne",
"Factors":{"Group":"FFAT(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_004_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_005_ne",
"Factors":{"Group":"Mock"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_005_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_006_ne",
"Factors":{"Group":"Mock(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_006_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_011_ne",
"Factors":{"Group":"Mock(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_011_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_007_ne",
"Factors":{"Group":"FL(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_007_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_008_ne",
"Factors":{"Group":"FFAT(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_008_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_012_ne",
"Factors":{"Group":"FFAT(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_012_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_009_ne",
"Factors":{"Group":"Mock"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_009_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"Mito_010_ne",
"Factors":{"Group":"FL(+)"},
"Additional sample data":{"RAW_FILE_NAME":"Mito_010_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"QC_mito_01_ne",
"Factors":{"Group":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_mito_01_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"QC_mito_02_ne",
"Factors":{"Group":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_mito_02_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"QC_mito_03_ne",
"Factors":{"Group":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_mito_03_ne.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"tQC_mito_03_ne",
"Factors":{"Group":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"tQC_mito_03_ne.mzml"}
}
],
"COLLECTION":{"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_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."},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"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.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters Acquity I-Class","COLUMN_NAME":"Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"ABI Sciex 5600 TripleTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","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.","MS_RESULTS_FILE":"ST001717_AN002797_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes"}

}