#METABOLOMICS WORKBENCH purdylab_20221230_115930 DATATRACK_ID:3678 STUDY_ID:ST002421 ANALYSIS_ID:AN003943 PROJECT_ID:PR001559 VERSION 1 CREATED_ON January 3, 2023, 10:19 am #PROJECT PR:PROJECT_TITLE UBXD8 lipidomics from whole cells PR:PROJECT_SUMMARY The intimate association between the endoplasmic reticulum (ER) and PR:PROJECT_SUMMARY mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a PR:PROJECT_SUMMARY platform for several critical cellular processes, in particular lipid synthesis. PR:PROJECT_SUMMARY Enzymes involved in lipid biosynthesis are enriched at contacts and membrane PR:PROJECT_SUMMARY lipid composition at contacts is distinct relative to surrounding membranes. How PR:PROJECT_SUMMARY contacts are remodeled and the subsequent biological consequences of altered PR:PROJECT_SUMMARY contacts such as perturbed lipid metabolism remains poorly understood. Here we PR:PROJECT_SUMMARY investigate if the ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulates PR:PROJECT_SUMMARY the lipidome of cells. LC-MS/MS lipidomics found significant changes in distinct PR:PROJECT_SUMMARY lipid species in UBXD8 knockout cells, in particular in saturated or PR:PROJECT_SUMMARY mono-unsaturated lipid species. Perturbation of contacts and inherent lipid PR:PROJECT_SUMMARY synthesis is emerging as a hallmark in a variety of human disorders such as PR:PROJECT_SUMMARY neurodegeneration. Our results suggest that contacts are exquisitely sensitive PR:PROJECT_SUMMARY to alterations to membrane lipid composition and saturation in a manner that is PR:PROJECT_SUMMARY dependent on UBXD8. PR:INSTITUTE University of Arizona PR:DEPARTMENT Immunobiology PR:LABORATORY Purdy Lab PR:LAST_NAME Purdy PR:FIRST_NAME John PR:ADDRESS PO Box 245221, Tucson, Arizona, 85724, USA PR:EMAIL purdylab@gmail.com PR:PHONE 520-626-4371 PR:FUNDING_SOURCE NIH R01 AI162671 PR:CONTRIBUTORS Rakesh Ganji, Joao A. Paulo, Yuecheng Xi, Ian Kline, Jiang Zhu, Christoph S. PR:CONTRIBUTORS Clemen, Conrad C. Weihl, John G. Purdy, Steve P. Gygi, and Malavika Raman #STUDY ST:STUDY_TITLE UBXD8 lipidomics from whole cells (Part 1) ST:STUDY_SUMMARY The intimate association between the endoplasmic reticulum (ER) and ST:STUDY_SUMMARY mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a ST:STUDY_SUMMARY platform for several critical cellular processes, in particular lipid synthesis. ST:STUDY_SUMMARY Enzymes involved in lipid biosynthesis are enriched at contacts and membrane ST:STUDY_SUMMARY lipid composition at contacts is distinct relative to surrounding membranes. How ST:STUDY_SUMMARY contacts are remodeled and the subsequent biological consequences of altered ST:STUDY_SUMMARY contacts such as perturbed lipid metabolism remains poorly understood. Here we ST:STUDY_SUMMARY investigate if the ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulates ST:STUDY_SUMMARY the lipidome of cells. LC-MS/MS lipidomics found significant changes in distinct ST:STUDY_SUMMARY lipid species in UBXD8 knockout cells, in particular in saturated or ST:STUDY_SUMMARY mono-unsaturated lipid species. Perturbation of contacts and inherent lipid ST:STUDY_SUMMARY synthesis is emerging as a hallmark in a variety of human disorders such as ST:STUDY_SUMMARY neurodegeneration. Our results suggest that contacts are exquisitely sensitive ST:STUDY_SUMMARY to alterations to membrane lipid composition and saturation in a manner that is ST:STUDY_SUMMARY dependent on UBXD8. ST:INSTITUTE University of Arizona ST:DEPARTMENT Immunobiology ST:LABORATORY Purdy Lab ST:LAST_NAME Purdy ST:FIRST_NAME John ST:ADDRESS PO Box 245221, Tucson, Arizona, 85724, USA ST:EMAIL purdylab@gmail.com ST:PHONE 520-626-4371 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS HEK293T #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 - 20190829_neg_KO_1 Description:UBXD8 Knockout RAW_FILE_NAME=20190829_neg_KO_1.mzXML SUBJECT_SAMPLE_FACTORS - 20190829_neg_KO_2 Description:UBXD8 Knockout RAW_FILE_NAME=20190829_neg_KO_2.mzXML SUBJECT_SAMPLE_FACTORS - 20190829_neg_wt_1 Description:Wild-type RAW_FILE_NAME=20190829_neg_wt_1.mzXML SUBJECT_SAMPLE_FACTORS - 20190829_neg_wt_2 Description:Wild-type RAW_FILE_NAME=20190829_neg_wt_2.mzXML SUBJECT_SAMPLE_FACTORS - 20190829_pos_KO_1 Description:UBXD8 Knockout RAW_FILE_NAME=20190829_pos_KO_1.mzXML SUBJECT_SAMPLE_FACTORS - 20190829_pos_KO_2 Description:UBXD8 Knockout RAW_FILE_NAME=20190829_pos_KO_2.mzXML SUBJECT_SAMPLE_FACTORS - 20190829_pos_wt_1 Description:Wild-type RAW_FILE_NAME=20190829_pos_wt_1.mzXML SUBJECT_SAMPLE_FACTORS - 20190829_pos_wt_2 Description:Wild-type RAW_FILE_NAME=20190829_pos_wt_2.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_neg_rep2_KO_a Description:UBXD8 Knockout RAW_FILE_NAME=20191130_neg_rep2_KO_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_neg_rep2_KO_b Description:UBXD8 Knockout RAW_FILE_NAME=20191130_neg_rep2_KO_b.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_neg_rep2_WT_a Description:Wild-type RAW_FILE_NAME=20191130_neg_rep2_WT_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_neg_rep2_WT_b Description:Wild-type RAW_FILE_NAME=20191130_neg_rep2_WT_b.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_neg_rep3_KO_a Description:UBXD8 Knockout RAW_FILE_NAME=20191130_neg_rep3_KO_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_neg_rep3_KO_b Description:UBXD8 Knockout RAW_FILE_NAME=20191130_neg_rep3_KO_b.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_neg_rep3_WT_a Description:Wild-type RAW_FILE_NAME=20191130_neg_rep3_WT_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_neg_rep3_WT_b Description:Wild-type RAW_FILE_NAME=20191130_neg_rep3_WT_b.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_pos_rep2_KO_a Description:UBXD8 Knockout RAW_FILE_NAME=20191130_pos_rep2_KO_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_pos_rep2_KO_b Description:UBXD8 Knockout RAW_FILE_NAME=20191130_pos_rep2_KO_b.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_pos_rep2_WT_a Description:Wild-type RAW_FILE_NAME=20191130_pos_rep2_WT_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_pos_rep2_WT_b Description:Wild-type RAW_FILE_NAME=20191130_pos_rep2_WT_b.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_pos_rep3_KO_a Description:UBXD8 Knockout RAW_FILE_NAME=20191130_pos_rep3_KO_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_pos_rep3_KO_b Description:UBXD8 Knockout RAW_FILE_NAME=20191130_pos_rep3_KO_b.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_pos_rep3_WT_a Description:Wild-type RAW_FILE_NAME=20191130_pos_rep3_WT_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191130_pos_rep3_WT_b Description:Wild-type RAW_FILE_NAME=20191130_pos_rep3_WT_b.mzXML SUBJECT_SAMPLE_FACTORS - 20191230_neg_rep2_KO_a Description:UBXD8 Knockout RAW_FILE_NAME=20191230_neg_rep2_KO_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191230_neg_rep2_KO_b Description:UBXD8 Knockout RAW_FILE_NAME=20191230_neg_rep2_KO_b.mzXML SUBJECT_SAMPLE_FACTORS - 20191230_neg_rep2_WT_a Description:Wild-type RAW_FILE_NAME=20191230_neg_rep2_WT_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191230_neg_rep2_WT_b Description:Wild-type RAW_FILE_NAME=20191230_neg_rep2_WT_b.mzXML SUBJECT_SAMPLE_FACTORS - 20191230_pos_rep2_KO_a Description:UBXD8 Knockout RAW_FILE_NAME=20191230_pos_rep2_KO_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191230_pos_rep2_KO_b Description:UBXD8 Knockout RAW_FILE_NAME=20191230_pos_rep2_KO_b.mzXML SUBJECT_SAMPLE_FACTORS - 20191230_pos_rep2_WT_a Description:Wild-type RAW_FILE_NAME=20191230_pos_rep2_WT_a.mzXML SUBJECT_SAMPLE_FACTORS - 20191230_pos_rep2_WT_b Description:Wild-type RAW_FILE_NAME=20191230_pos_rep2_WT_b.mzXML #COLLECTION CO:COLLECTION_SUMMARY Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), CO:COLLECTION_SUMMARY supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and CO:COLLECTION_SUMMARY streptomycin. Cells were maintained in a humidified, 5 % CO2 atmosphere at CO:COLLECTION_SUMMARY 37°C. The CRISPR-Cas9 gene editing system was used to generate UBXD8 knockout CO:COLLECTION_SUMMARY cell lines in HEK293T cells. Cells were grown in 6-well plates for lipidomics. CO:COLLECTION_SUMMARY Cells were washed with PBS, scraped into cold 50% methanol, centrifuged, and the CO:COLLECTION_SUMMARY cell pellets were frozen at -80˚C. Next, cells were resuspended in cold 50% CO:COLLECTION_SUMMARY methanol (1mL) and transferred to glass vials. Chloroform was added (0.5mL) and CO:COLLECTION_SUMMARY the mixture was gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. CO:COLLECTION_SUMMARY Lipids were transferred to a clean glass vial using a glass Hamilton syringe. CO:COLLECTION_SUMMARY Lipids were extracted twice using chloroform prior to being dried under nitrogen CO:COLLECTION_SUMMARY gas. Samples were normalized according to protein concentration when resuspended CO:COLLECTION_SUMMARY in a 1:1:1 solution of methanol:chloroform:isopropanol prior to mass CO:COLLECTION_SUMMARY spectrometry (MS) analysis. CO:SAMPLE_TYPE Epithelial cells #TREATMENT TR:TREATMENT_SUMMARY Wild-type vs UBXD8 Knockout #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipids were isolated from collected cultured cells. Cells were washed with PBS, SP:SAMPLEPREP_SUMMARY treated with cold 50% methanol (1mL) and transferred to glass vials. Next, SP:SAMPLEPREP_SUMMARY chloroform (0.5mL) was added and samples were gently vortexed and centrifuged at SP:SAMPLEPREP_SUMMARY 1,000x g for 5 min at 4˚C. Lipids were transferred to a clean glass vial using SP:SAMPLEPREP_SUMMARY a glass Hamilton syringe. Lipids were extracted twice using chloroform prior to SP:SAMPLEPREP_SUMMARY being dried under nitrogen gas. Samples were normalized according to protein SP:SAMPLEPREP_SUMMARY concentration when resuspended in a 1:1:1 solution of SP:SAMPLEPREP_SUMMARY methanol:chloroform:isopropanol prior to mass spectrometry (MS) analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) CH:SOLVENT_A 40% water; 60% methanol 10mM ammonium formate and 0.1% formic acid CH:SOLVENT_B 10% methanol; 90% isopropanol 10mM ammonium formate and 0.1% formic acid CH:FLOW_GRADIENT 25% to 100% CH:FLOW_RATE 0.25mL per min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Samples were run in a semi-random order where WT or UBXD8 KO samples were MS:MS_COMMENTS interspersed with blank samples. Lipids were ionized using a heated electrospray MS:MS_COMMENTS ionization (HESI) source and nitrogen gas and measured using a Q-Exactive Plus MS:MS_COMMENTS mass spectrometer operating at a MS1 resolution of either 70,000 or 140,000 and MS:MS_COMMENTS a MS2 resolution of 35,000. MS1 Spectra were collected over a mass range of 200 MS:MS_COMMENTS to 1,600 m/z with an automatic gain control (AGC) setting of 1e6 and transient MS:MS_COMMENTS times of 250 ms (70,000 resolution) or 520 ms (140,000 resolution). MS2 spectra MS:MS_COMMENTS were collected using a transient time of 120 ms and an AGC setting of 1e5. Each MS:MS_COMMENTS sample was analyzed using negative and positive ion modes. The mass analyzer was MS:MS_COMMENTS calibrated weekly. SPLASH LIPIDOMIX mass spectrometry standards (Avanti Polar MS:MS_COMMENTS Lipids) were used in determining extraction efficiencies and lipid quantitation. MS:MS_COMMENTS Lipids were identified and quantified using MAVEN, and EI-MAVEN (Elucidata). MS:MS_COMMENTS UHPLC retention time, MS1 peaks, and MS2 fragments were used to identify lipids. MS:MS_COMMENTS Lipids were included if they were observed in 3-6 samples in both UBXD8 KO and MS:MS_COMMENTS WT cells. Missing values in a sample were not imputed. The following lipid MS:MS_COMMENTS classes were included in the analysis: cholesteryl esters (CE), diacylglycerol MS:MS_COMMENTS (DG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), MS:MS_COMMENTS phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), MS:MS_COMMENTS and triacylglycerol (TG). Guidelines from the Lipidomic Standards Initiative MS:MS_COMMENTS were followed for lipid species identification and quantification, including MS:MS_COMMENTS consideration of isotopic patterns resulting from naturally occurring 13C atoms MS:MS_COMMENTS and isomeric overlap. The following MS2 information was used to confirm each MS:MS_COMMENTS lipid species: PC fragment of 184.073 (positive mode) and tail identification MS:MS_COMMENTS using formic adduct (negative mode); PE fragment of 196.038 or the tail plus MS:MS_COMMENTS 197.046 (negative mode) and neutral loss (NL) of 141.019 (positive mode); PG MS:MS_COMMENTS fragment of 152.996 plus the identification of the FA tails (negative mode) and MS:MS_COMMENTS NL 189.04 of [M+NH4]+ adduct (positive mode); PI fragment of 241.012 (negative) MS:MS_COMMENTS and NL 277.056 of [M+NH4]+ adduct (positive mode); PS NL of 87.032 (negative); MS:MS_COMMENTS DG and TG by NL of FA tails (positive mode); and CE fragment of 369.352 or MS:MS_COMMENTS neutral loss of 368.35 (positive). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples 20190829_neg_wt_1 20190829_neg_wt_2 20190829_neg_KO_1 20190829_neg_KO_2 20191130_neg_rep2_WT_a 20191130_neg_rep2_WT_b 20191130_neg_rep2_KO_a 20191130_neg_rep2_KO_b 20191130_neg_rep3_WT_a 20191130_neg_rep3_WT_b 20191130_neg_rep3_KO_a 20191130_neg_rep3_KO_b 20191230_neg_rep2_WT_a 20191230_neg_rep2_WT_b 20191230_neg_rep2_KO_a 20191230_neg_rep2_KO_b Factors Description:Wild-type Description:Wild-type Description:UBXD8 Knockout Description:UBXD8 Knockout Description:Wild-type Description:Wild-type Description:UBXD8 Knockout Description:UBXD8 Knockout Description:Wild-type Description:Wild-type Description:UBXD8 Knockout Description:UBXD8 Knockout Description:Wild-type Description:Wild-type Description:UBXD8 Knockout Description:UBXD8 Knockout PS(34:1) 971658.5 1756208 1525648 1507113 559238.69 720422.5 461804.72 480223.88 101659.06 141150.61 141153.2 191344.92 106098 149494.02 151213.61 252006.94 PS(36:1) 2724608 4678279 5507016 4629795 1405889.5 1957755.62 1669307.38 1964916.88 401618.41 484730.09 733068 966368.06 314345.97 410583.78 611699.38 987552.25 PS(36:2) 465843.3 787087.5 576887.1 721140.6 149691.77 243593.55 198049.05 180617.27 24499.22 33650.46 39350.23 61810 37375.22 60248.7 79462.55 137512.92 PS(38:1) 507002 783698.8 792627.5 885821.1 144192.19 191772.38 177234.98 183983.67 45134.86 55910.38 89826.35 109059.68 34446.61 58761.71 69095.41 117153.1 PS(38:2) 379861.4 573755 425413 546686.4 74651.95 132591.5 100238.59 128563.34 18936.09 20245.65 24925.23 38104.2 20365.94 32171.57 45465.48 70470.54 PS(38:4) 137203.4 439868.5 265522.1 758992.6 47678.36 69070.48 53352.32 50594.16 12170.4 13631.89 12051.15 17922.36 14885.78 17119.54 17863.06 37079.73 PS(40:1) 1716493 2607646 2297049 2008633 290384.28 330682.34 212324.52 217504.5 38162.34 42892.73 92056.84 107005 38848.38 41283.8 80856.48 136718.23 PS(40:4) 176897.6 242054.1 359139.2 416641.3 111025.04 141162.92 175008.14 189242.83 37653.5 43896.94 51039.5 68447.04 42490.18 58408.43 126810.45 196778.38 PS(40:6) 81906.75 112344.6 178615.5 222042.2 76129.2 99015.07 179118.8 180053.58 35438.77 50907.39 56314.25 72040.21 39451.79 60026.95 88996.79 139968.39 PS(42:1) 33805.58 1269440 1331618 2100709 76774.13 87624.37 98080.25 104207.31 11279.84 16428.11 48455.96 51909.12 13118.23 24590.16 51778.62 75269.16 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 20190829_neg_wt_1 20190829_neg_wt_2 20190829_neg_KO_1 20190829_neg_KO_2 20191130_neg_rep2_WT_a 20191130_neg_rep2_WT_b 20191130_neg_rep2_KO_a 20191130_neg_rep2_KO_b 20191130_neg_rep3_WT_a 20191130_neg_rep3_WT_b 20191130_neg_rep3_KO_a 20191130_neg_rep3_KO_b 20191230_neg_rep2_WT_a 20191230_neg_rep2_WT_b 20191230_neg_rep2_KO_a 20191230_neg_rep2_KO_b PS(34:1) 971658.5 1756208 1525648 1507113 559238.69 720422.5 461804.72 480223.88 101659.06 141150.61 141153.2 191344.92 106098 149494.02 151213.61 252006.94 PS(36:1) 2724608 4678279 5507016 4629795 1405889.5 1957755.62 1669307.38 1964916.88 401618.41 484730.09 733068 966368.06 314345.97 410583.78 611699.38 987552.25 PS(36:2) 465843.3 787087.5 576887.1 721140.6 149691.77 243593.55 198049.05 180617.27 24499.22 33650.46 39350.23 61810 37375.22 60248.7 79462.55 137512.92 PS(38:1) 507002 783698.8 792627.5 885821.1 144192.19 191772.38 177234.98 183983.67 45134.86 55910.38 89826.35 109059.68 34446.61 58761.71 69095.41 117153.1 PS(38:2) 379861.4 573755 425413 546686.4 74651.95 132591.5 100238.59 128563.34 18936.09 20245.65 24925.23 38104.2 20365.94 32171.57 45465.48 70470.54 PS(38:4) 137203.4 439868.5 265522.1 758992.6 47678.36 69070.48 53352.32 50594.16 12170.4 13631.89 12051.15 17922.36 14885.78 17119.54 17863.06 37079.73 PS(40:1) 1716493 2607646 2297049 2008633 290384.28 330682.34 212324.52 217504.5 38162.34 42892.73 92056.84 107005 38848.38 41283.8 80856.48 136718.23 PS(40:4) 176897.6 242054.1 359139.2 416641.3 111025.04 141162.92 175008.14 189242.83 37653.5 43896.94 51039.5 68447.04 42490.18 58408.43 126810.45 196778.38 PS(40:6) 81906.75 112344.6 178615.5 222042.2 76129.2 99015.07 179118.8 180053.58 35438.77 50907.39 56314.25 72040.21 39451.79 60026.95 88996.79 139968.39 PS(42:1) 33805.58 1269440 1331618 2100709 76774.13 87624.37 98080.25 104207.31 11279.84 16428.11 48455.96 51909.12 13118.23 24590.16 51778.62 75269.16 METABOLITES_END #END