#METABOLOMICS WORKBENCH taonotky_20241201_010906 DATATRACK_ID:5414 STUDY_ID:ST003658 ANALYSIS_ID:AN006008 PROJECT_ID:PR002268 VERSION 1 CREATED_ON January 9, 2025, 1:16 am #PROJECT PR:PROJECT_TITLE Role of lipid-metabolizing enzyme PNPLA6 in retinal pigment epithelial cells and PR:PROJECT_TITLE its mechanism of homeostasis PR:PROJECT_SUMMARY PNPLA6 deficiency in the retina has been clinically reported to be a cause of PR:PROJECT_SUMMARY retinitis pigmentosa, a disease leading to blindness. PNPLA6 is generally known PR:PROJECT_SUMMARY to be an enzyme that degrades phospholipids in a calcium-independent manner, but PR:PROJECT_SUMMARY its activity and substrates in the eye have not yet been elucidated. We focused PR:PROJECT_SUMMARY on the function of PNPLA6, whose function in the retina is not well understood, PR:PROJECT_SUMMARY and biochemically analyzed the mechanism by which retinal pigmentary PR:PROJECT_SUMMARY degeneration occurs. PR:INSTITUTE University of Tokyo PR:LAST_NAME Ono PR:FIRST_NAME Takashi PR:ADDRESS 7-3-1, Hongo, Bunkyo-ku PR:EMAIL taono-tky@umin.ac.jp PR:PHONE +81-338155411 #STUDY ST:STUDY_TITLE Functional analysis of retinal pigment epithelial cells with PNPLA6 knockdown ST:STUDY_SUMMARY PNPLA6 was knocked down using siRNA on ARPE-19, a representative cultured cell ST:STUDY_SUMMARY line of human retinal pigment epithelial cells. Lipid analysis of the ST:STUDY_SUMMARY constituent phospholipids was performed on these cells, comparing them to a ST:STUDY_SUMMARY group treated with scramble siRNA. We also overexpressed PNPLA6 in human retinal ST:STUDY_SUMMARY pigment epithelial cells ARPE-19 and performed the same lipid analysis. ST:INSTITUTE University of Tokyo ST:LAST_NAME Ono ST:FIRST_NAME Takashi ST:ADDRESS 7-3-1, Hongo, Bunkyo-ku ST:EMAIL taono-tky@umin.ac.jp ST:PHONE 0338155411 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS n1 NegKD-1 Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-n1.mzML SUBJECT_SAMPLE_FACTORS n2 NegKD-2 Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-n2.mzML SUBJECT_SAMPLE_FACTORS n3 NegKD-3 Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-n3.mzML SUBJECT_SAMPLE_FACTORS n4 NegKD-4 Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-n4.mzML SUBJECT_SAMPLE_FACTORS 6kd1 PN6KD-1 Sample source:RPE (Retinal pigment epithelium) | Genotype:KD Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6kd1.mzML SUBJECT_SAMPLE_FACTORS 6kd2 PN6KD-2 Sample source:RPE (Retinal pigment epithelium) | Genotype:KD Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6kd2.mzML SUBJECT_SAMPLE_FACTORS 6kd3 PN6KD-3 Sample source:RPE (Retinal pigment epithelium) | Genotype:KD Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6kd3.mzML SUBJECT_SAMPLE_FACTORS 6kd4 PN6KD-4 Sample source:RPE (Retinal pigment epithelium) | Genotype:KD Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6kd4.mzML SUBJECT_SAMPLE_FACTORS mock1 mock-1 Sample source:RPE (Retinal pigment epithelium) | Genotype:mock Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-mock1.mzML SUBJECT_SAMPLE_FACTORS mock2 mock-2 Sample source:RPE (Retinal pigment epithelium) | Genotype:mock Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-mock2.mzML SUBJECT_SAMPLE_FACTORS mock3 mock-3 Sample source:RPE (Retinal pigment epithelium) | Genotype:mock Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-mock3.mzML SUBJECT_SAMPLE_FACTORS mock4 mock-4 Sample source:RPE (Retinal pigment epithelium) | Genotype:mock Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-mock4.mzML SUBJECT_SAMPLE_FACTORS 6OE1 PN6OE-1 Sample source:RPE (Retinal pigment epithelium) | Genotype:OE Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6OE1.mzML SUBJECT_SAMPLE_FACTORS 6OE2 PN6OE-2 Sample source:RPE (Retinal pigment epithelium) | Genotype:OE Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6OE2.mzML SUBJECT_SAMPLE_FACTORS 6OE3 PN6OE-3 Sample source:RPE (Retinal pigment epithelium) | Genotype:OE Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6OE3.mzML SUBJECT_SAMPLE_FACTORS 6OE4 PN6OE-4 Sample source:RPE (Retinal pigment epithelium) | Genotype:OE Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6OE4.mzML #COLLECTION CO:COLLECTION_SUMMARY The human RPE cell line ARPE-19 (CRL-2302, ATCC) was cultured in Dulbecco's CO:COLLECTION_SUMMARY Modified Eagle's Medium (DMEM)/F12 and DMEM high glucose , respectively, with CO:COLLECTION_SUMMARY penicillin streptomycin (100 units/ml) and 10% (v/v) fetal bovine serum in a CO2 CO:COLLECTION_SUMMARY incubator under 5% (v/v) CO2 at 37°C. Target-specific Silencer® Select siRNAs CO:COLLECTION_SUMMARY (Thermo Fischer Scientific) or a Silencer® Select Negative Control No. 1 siRNA CO:COLLECTION_SUMMARY (4390843, Thermo Fischer Scientific) were transfected into ARPE-19 with CO:COLLECTION_SUMMARY Lipofectamine 3000. Cells were collected once after transfection, re-spread, and CO:COLLECTION_SUMMARY collected 3 days later. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Cells were stripped using trypsin EDTA, washed with PBS, and cell counts were TR:TREATMENT_SUMMARY performed with automated cell counter. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Total lipids were extracted using the Bligh and Dyer method. After the samples SP:SAMPLEPREP_SUMMARY were dried, 175 µL of digestion reagent (60% HClO: H2SO4 = 1:1 (v/v)) was added SP:SAMPLEPREP_SUMMARY to the samples or a phosphorus standard (NaH2PO4), stirred, and scorched using a SP:SAMPLEPREP_SUMMARY gas burner. After cooling at room temperature, 125 µL of water, 1 mL of 1% SP:SAMPLEPREP_SUMMARY (w/v) ammonium molybdate, and 50 µL of reducing reagent were added and the SP:SAMPLEPREP_SUMMARY samples were boiled for 10 min. The reaction was stopped on ice, and the SP:SAMPLEPREP_SUMMARY absorbance was measured at 750 nm. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME LC-30AD CH:COLUMN_NAME SeQuant ZIC-HILIC (100 x 2.1 mm, 3.5 µm) CH:SOLVENT_A 95% Acetonitrile/5% Water; 10 mM ammonium acetate CH:SOLVENT_B 50% Acetonitrile/50% Water; 20 mM ammonium acetate CH:FLOW_GRADIENT 0–3 min 0% B; 3–6 min 0–60% B; 6–17 min 60–62.2% B; 17–17.01 min CH:FLOW_GRADIENT 62.2–69.8% B; 17.01–20 min 69.8–100% B; 20–23 min 100% B; 23–23.10 min CH:FLOW_GRADIENT 100–0% B; 23.10–26 min 0% B CH:FLOW_RATE 0.2 mL/min CH:COLUMN_TEMPERATURE 50°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 4000 QTrap MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE API MS:ION_MODE POSITIVE MS:MS_COMMENTS 4500 QTRAP LC-MS/MS/MS system (AB Sciex), a triple quadrupole lipid molecular MS:MS_COMMENTS apparatus, was employed to quantify individual lipid molecules. Lipid samples (5 MS:MS_COMMENTS nmol of phospholipids) or standard lipids (Avanti Polar Lipids), prepared as MS:MS_COMMENTS previously described, were transferred to vials with added internal standards MS:MS_COMMENTS for each lipid type. The solvent was then evaporated using nitrogen gas, and the MS:MS_COMMENTS residue was dissolved in 50 µL of isopropanol:methanol (1:1, v/v) before being MS:MS_COMMENTS placed in an autosampler. Lipid profiling was conducted using a concurrent MS:MS_COMMENTS quantification method for phospholipids and lysophospholipids via multiple MS:MS_COMMENTS reaction monitoring (MRM). MultiQuant software (AB Sciex) was utilized to MS:MS_COMMENTS analyze the data, yielding peak area under the curve (AUC) values for each lipid MS:MS_COMMENTS molecule. Quantification of individual lipid species was achieved using MS:MS_COMMENTS calibration curves generated from AUCs obtained at LC elution times matching MS:MS_COMMENTS those of the standard lipids. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/10^6 cells MS_METABOLITE_DATA_START Samples NegKD-1 NegKD-2 NegKD-3 NegKD-4 PN6KD-1 PN6KD-2 PN6KD-3 PN6KD-4 Factors Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Sample source:RPE (Retinal pigment epithelium) | Genotype:KD Sample source:RPE (Retinal pigment epithelium) | Genotype:KD Sample source:RPE (Retinal pigment epithelium) | Genotype:KD Sample source:RPE (Retinal pigment epithelium) | Genotype:KD LPC 16:0 4901678.002 4354088.779 4977222.81 4193371.477 6244821.809 5941081.084 5088736.799 5831746.468 LPC 16:1 391015.2988 312431.0667 370566.7106 340505.271 520395.7498 421152.8802 407764.7858 455266.9705 LPC 17:0 361023.5445 309166.9209 372762.9602 312842.2384 533606.1016 444928.3585 437690.1149 478295.8901 LPC 18:0 4879619.882 4270381.329 5324235.837 4303610.17 7192406.563 5711670.64 5518903.408 6227195.453 LPC 18:1 3179939.122 2600375.747 3024300.528 2699631.253 4654169.006 3881783.299 3450029.822 3902567.557 LPC 18:2 303397.7921 277909.7473 352297.3056 282646.0848 419705.6598 368345.2495 283033.2545 368981.2676 LPC 18:3 17148.93913 14149.84147 12332.93607 10873.27235 19855.6308 15371.68239 20690.42363 16896.64053 LPC 20:0 110836.7341 107055.5117 124182.2355 109078.5376 173695.6094 132921.29 137931.6453 146186.4789 LPC 20:1 176649.9745 135928.1705 153604.716 167664.847 235154.1166 181273.5184 170923.7694 185243.7437 LPC 20:2 99084.92204 88232.0957 96006.50147 84802.77245 126888.2433 98906.85297 83733.47007 94263.71603 LPC 20:3 357467.6422 258016.9798 314922.9483 331376.3205 501332.2239 393848.7752 355324.499 442286.5995 LPC 20:4 579093.4202 475792.5751 549215.3172 491047.3665 779347.1051 660815.8869 672629.1808 740027.1422 LPC 20:5 40895.93418 25099.23043 29230.33624 20133.28707 67008.74654 45840.21171 53480.23054 36230.81547 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention index m/z LPC 16:0 10.73212685 496.3 LPC 16:1 10.74750604 494.3 LPC 17:0 10.77570914 510.3 LPC 18:0 10.59116749 524.3 LPC 18:1 10.50917107 522.3 LPC 18:2 10.79771404 520.3 LPC 18:3 10.93419458 518.3 LPC 20:0 10.59108428 552.3 LPC 20:1 10.59759584 550.3 LPC 20:2 10.5230088 548.3 LPC 20:3 10.8629894 546.3 LPC 20:4 10.6031658 544.3 LPC 20:5 10.5644 542.3 METABOLITES_END #END