#METABOLOMICS WORKBENCH taonotky_20241230_005946 DATATRACK_ID:5496 STUDY_ID:ST003648 ANALYSIS_ID:AN005994 PROJECT_ID:PR002258 VERSION 1 CREATED_ON January 2, 2025, 10:01 pm #PROJECT PR:PROJECT_TITLE Function of retinal pigment epithelium in the absence of PNPLA6 PR:PROJECT_SUMMARY Deletion of PNPLA6 has been reported to cause retinitis pigmentosa with PR:PROJECT_SUMMARY neurodegeneration.Since PNPLA6 is predominantly expressed in retinal pigment PR:PROJECT_SUMMARY epithelial cells in the retina, in this study PNPLA6 was knocked down in retinal PR:PROJECT_SUMMARY pigment epithelial cells and its lipid composition was analysed. 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 Analysis of the role of PNPLA6 in the mouse retina ST:STUDY_SUMMARY We generated PNPLA6 flox/flox CreER(+) mice, knocked out PNPLA6 by treatment ST:STUDY_SUMMARY with tamoxifen and isolated their retinal pigment epithelium. Lipid analysis of ST:STUDY_SUMMARY the retinal pigment epithelial cells was subsequently performed. 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 Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 control1 C-1 Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-c1.mzML SUBJECT_SAMPLE_FACTORS control2 C-2 Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-c2.mzML SUBJECT_SAMPLE_FACTORS control3 C-3 Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-c3.mzML SUBJECT_SAMPLE_FACTORS control4 C-4 Sample source:RPE (Retinal pigment epithelium) | Genotype:Wild Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-c4.mzML SUBJECT_SAMPLE_FACTORS 6ko1 PN6KO-1 Sample source:RPE (Retinal pigment epithelium) | Genotype:KO Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6ko1.mzML SUBJECT_SAMPLE_FACTORS 6ko2 PN6KO-2 Sample source:RPE (Retinal pigment epithelium) | Genotype:KO Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6ko2.mzML SUBJECT_SAMPLE_FACTORS 6ko3 PN6KO-3 Sample source:RPE (Retinal pigment epithelium) | Genotype:KO Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6ko3.mzML SUBJECT_SAMPLE_FACTORS 6ko4 PN6KO-4 Sample source:RPE (Retinal pigment epithelium) | Genotype:KO Treatment=-; Batch=B1a; RAW_FILE_NAME(RAW_file_name)=MS-6ko4.mzML #COLLECTION CO:COLLECTION_SUMMARY We established PNPLA6 flox/flox CreER(+) mice by mating and knocking out PNPLA6 CO:COLLECTION_SUMMARY by treatment with tamoxifen. PNPLA6 flox/flox CreER(-) mice were used as a CO:COLLECTION_SUMMARY control group. The eye was excised, the corneal ring was incised under a CO:COLLECTION_SUMMARY microscope, the retina was removed and the retinal pigment epithelium was CO:COLLECTION_SUMMARY isolated bluntly. The isolated retinal pigment epithelial cells were analyzed CO:COLLECTION_SUMMARY for lipids using LC-MS/MS. CO:SAMPLE_TYPE Eye tissue #TREATMENT TR:TREATMENT_SUMMARY After Pnpla6fl/flCAG-CreER (+) and Pnpla6fl/flCAG-CreER (-) were euthanized by TR:TREATMENT_SUMMARY cervical dislocation, the eyes were isolated, periocular connective tissue was TR:TREATMENT_SUMMARY removed, and the optic nerve was isolated. The cornea was separated at the TR:TREATMENT_SUMMARY corneal ring using a Vannas shear (and then the iris and lens were removed. An TR:TREATMENT_SUMMARY intact RPE sheet was removed from the underlying Bruch's membrane using a TR:TREATMENT_SUMMARY microsurgical crescent knife under a dissecting microscope. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Retinal pigment epithelium was collected from the retina with microscope. After SP:SAMPLEPREP_SUMMARY two washes with culture medium, retinal pigment epithelial cells were carefully SP:SAMPLEPREP_SUMMARY collected in 1.5 mL centrifuge tubes and carefully resuspended using SP:SAMPLEPREP_SUMMARY micropipette, avoiding contamination of other tissues such as retinal debris and SP:SAMPLEPREP_SUMMARY choroid. #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 C-1 C-2 C-3 C-4 PN6KO-1 PN6KO-2 PN6KO-3 PN6KO-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:KO Sample source:RPE (Retinal pigment epithelium) | Genotype:KO Sample source:RPE (Retinal pigment epithelium) | Genotype:KO Sample source:RPE (Retinal pigment epithelium) | Genotype:KO LPC 16:0 0.077574768 0.07288051 0.07029789 0.084329413 0.057101309 0.049448014 0.064715251 0.045029556 LPC 18:0 0.062548415 0.059356362 0.058393512 0.070574559 0.052729595 0.047574301 0.041384331 LPC 18:1 0.020017598 0.01898331 0.018152766 0.022456259 0.012986742 0.011934369 0.017823245 0.011022833 LPC 18:2 0.01606369 0.015387413 0.012884655 0.017434395 0.012668799 0.011298545 0.013635082 0.008761871 LPC 18:3 3.72013E-05 3.95686E-05 3.65806E-05 0.000307313 3.96328E-06 0.00012829 1.22048E-05 0.000163446 LPC 20:4 0.006239808 0.00747369 0.006167286 0.006642915 0.003998743 0.003794128 0.005373524 0.003344789 LPC 20:5 0.000727038 0.000503907 0.000687393 0.00066216 0.000463341 0.000396237 0.000376592 0.000503 LPC 22:5 0.001146333 0.00119359 0.001103694 0.001147291 0.000793023 0.000573367 0.001057747 0.000522431 LPC 22:6 0.010147862 0.008326254 0.009887404 0.011757162 0.004715337 0.005490035 0.006082685 0.00414356 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z LPC 16:0 10.73212685 496.3 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 20:4 10.6031658 544.3 LPC 20:5 10.5644 542.3 LPC 22:5 10.47639134 570.2 LPC 22:6 10.57170707 568.2 METABOLITES_END #END