#METABOLOMICS WORKBENCH purdylab_20230101_124221 DATATRACK_ID:3679 STUDY_ID:ST002422 ANALYSIS_ID:AN003945 PROJECT_ID:PR001559 VERSION 1 CREATED_ON January 3, 2023, 10:19 am #PROJECT PR:PROJECT_TITLE UBXD8 lipidomics from MAM fraction 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 lipids found in mitochondria-associated membranes (MAM). LC-MS/MS lipidomics PR:PROJECT_SUMMARY found significant changes in distinct lipid species in the MAM fraction of UBXD8 PR:PROJECT_SUMMARY knockout cells. Our results suggest that lipids in MAM are regulated by 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 2) 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 lipids found in mitochondria-associated membranes (MAM). LC-MS/MS lipidomics ST:STUDY_SUMMARY found significant changes in distinct lipid species in the MAM fraction of UBXD8 ST:STUDY_SUMMARY knockout cells. Our results suggest that lipids in MAM are regulated by 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 - 20220508_neg_KO_rep1_10ul Description:UBXD8 Knockout | inj vol:10 RAW_FILE_NAME=20220508_neg_KO_rep1_10ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_neg_KO_rep3_10ul Description:UBXD8 Knockout | inj vol:10 RAW_FILE_NAME=20220508_neg_KO_rep3_10ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_neg_KO_rep4_10ul Description:UBXD8 Knockout | inj vol:10 RAW_FILE_NAME=20220508_neg_KO_rep4_10ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_neg_WT_rep1_10ul Description:Wild-type | inj vol:10 RAW_FILE_NAME=20220508_neg_WT_rep1_10ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_neg_WT_rep3_10ul Description:Wild-type | inj vol:10 RAW_FILE_NAME=20220508_neg_WT_rep3_10ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_neg_WT_rep4_10ul Description:Wild-type | inj vol:10 RAW_FILE_NAME=20220508_neg_WT_rep4_10ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_KO_rep1_4ul Description:UBXD8 Knockout | inj vol:4 RAW_FILE_NAME=20220508_pos_KO_rep1_4ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_KO_rep1_8ul Description:UBXD8 Knockout | inj vol:8 RAW_FILE_NAME=20220508_pos_KO_rep1_8ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_KO_rep1_16ul Description:UBXD8 Knockout | inj vol:16 RAW_FILE_NAME=20220508_pos_KO_rep1_16ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_KO_rep3_4ul Description:UBXD8 Knockout | inj vol:4 RAW_FILE_NAME=20220508_pos_KO_rep3_4ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_KO_rep3_8ul Description:UBXD8 Knockout | inj vol:8 RAW_FILE_NAME=20220508_pos_KO_rep3_8ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_KO_rep3_16ul Description:UBXD8 Knockout | inj vol:16 RAW_FILE_NAME=20220508_pos_KO_rep3_16ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_KO_rep4_4ul Description:UBXD8 Knockout | inj vol:4 RAW_FILE_NAME=20220508_pos_KO_rep4_4ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_KO_rep4_8ul Description:UBXD8 Knockout | inj vol:8 RAW_FILE_NAME=20220508_pos_KO_rep4_8ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_KO_rep4_16ul Description:UBXD8 Knockout | inj vol:16 RAW_FILE_NAME=20220508_pos_KO_rep4_16ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_WT_rep1_4ul Description:Wild-type | inj vol:4 RAW_FILE_NAME=20220508_pos_WT_rep1_4ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_WT_rep1_8ul Description:Wild-type | inj vol:8 RAW_FILE_NAME=20220508_pos_WT_rep1_8ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_WT_rep1_16ul Description:Wild-type | inj vol:16 RAW_FILE_NAME=20220508_pos_WT_rep1_16ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_WT_rep3_4ul Description:Wild-type | inj vol:4 RAW_FILE_NAME=20220508_pos_WT_rep3_4ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_WT_rep3_8ul Description:Wild-type | inj vol:8 RAW_FILE_NAME=20220508_pos_WT_rep3_8ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_WT_rep3_16ul Description:Wild-type | inj vol:16 RAW_FILE_NAME=20220508_pos_WT_rep3_16ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_WT_rep4_4ul Description:Wild-type | inj vol:4 RAW_FILE_NAME=20220508_pos_WT_rep4_4ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_WT_rep4_8ul Description:Wild-type | inj vol:8 RAW_FILE_NAME=20220508_pos_WT_rep4_8ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220508_pos_WT_rep4_16ul Description:Wild-type | inj vol:16 RAW_FILE_NAME=20220508_pos_WT_rep4_16ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_neg_rep8_KO_5ul Description:UBXD8 Knockout | inj vol:5 RAW_FILE_NAME=20220820_tufts_MAMfrac_neg_rep8_KO_5ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_neg_rep8_KO_10ul Description:UBXD8 Knockout | inj vol:10 RAW_FILE_NAME=20220820_tufts_MAMfrac_neg_rep8_KO_10ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_neg_rep8_KO_15ul Description:UBXD8 Knockout | inj vol:15 RAW_FILE_NAME=20220820_tufts_MAMfrac_neg_rep8_KO_15ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_neg_rep8_WT_5ul Description:Wild-type | inj vol:5 RAW_FILE_NAME=20220820_tufts_MAMfrac_neg_rep8_WT_5ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_neg_rep8_WT_10ul Description:Wild-type | inj vol:10 RAW_FILE_NAME=20220820_tufts_MAMfrac_neg_rep8_WT_10ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_neg_rep8_WT_15ul Description:Wild-type | inj vol:15 RAW_FILE_NAME=20220820_tufts_MAMfrac_neg_rep8_WT_15ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_pos_rep8_KO_4ul Description:UBXD8 Knockout | inj vol:4 RAW_FILE_NAME=20220820_tufts_MAMfrac_pos_rep8_KO_4ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_pos_rep8_KO_8ul Description:UBXD8 Knockout | inj vol:8 RAW_FILE_NAME=20220820_tufts_MAMfrac_pos_rep8_KO_8ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_pos_rep8_KO_16ul Description:UBXD8 Knockout | inj vol:16 RAW_FILE_NAME=20220820_tufts_MAMfrac_pos_rep8_KO_16ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_pos_rep8_WT_4ul Description:Wild-type | inj vol:4 RAW_FILE_NAME=20220820_tufts_MAMfrac_pos_rep8_WT_4ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_pos_rep8_WT_8ul Description:Wild-type | inj vol:8 RAW_FILE_NAME=20220820_tufts_MAMfrac_pos_rep8_WT_8ul.mzXML SUBJECT_SAMPLE_FACTORS - 20220820_tufts_MAMfrac_pos_rep8_WT_16ul Description:Wild-type | inj vol:16 RAW_FILE_NAME=20220820_tufts_MAMfrac_pos_rep8_WT_16ul.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 seeded into four 150 mm TC dishes. Cells CO:COLLECTION_SUMMARY were lysed in Homogenization buffer (225 mM mannitol, 75 mM sucrose, and 30 mM CO:COLLECTION_SUMMARY Tris-Cl, pH 7.4) using a Dounce homogenizer. The lysate was centrifuged three CO:COLLECTION_SUMMARY times at 600xg for 5 minutes to remove unlysed cells and nuclei resulting in CO:COLLECTION_SUMMARY post-nuclear supernatants (PNS). The cleared lysate was centrifuged at 7000xg to CO:COLLECTION_SUMMARY separate crude mitochondrial pellet and supernatant containing microsomes. The CO:COLLECTION_SUMMARY supernatant was cleared by centrifugation at 20,000xg for 30 minutes followed by CO:COLLECTION_SUMMARY microsome isolation using high-speed centrifugation at 100,000xg for 1 hour. The CO:COLLECTION_SUMMARY crude mitochondrial pellet was washed twice in homogenization buffer containing CO:COLLECTION_SUMMARY 0.1 mM EGTA at 7000xg and 10,000xg for 10 minutes. MAMs were isolated from crude CO:COLLECTION_SUMMARY mitochondria using 30 % Percoll gradient centrifugation at 95,000xg for 1 hr in CO:COLLECTION_SUMMARY a swinging-bucket rotor. The banded MAM fraction was washed once with CO:COLLECTION_SUMMARY phosphate-buffered saline (PBS) before lysing in lysis buffer (50 mM Tris-Cl, pH CO:COLLECTION_SUMMARY 7.2, 150 mM NaCl). The pure mitochondrial fractions were resuspended and washed CO:COLLECTION_SUMMARY in mitochondrial resuspension buffer (250 mM mannitol, 0.5 mM EGTA, 5 mM HEPES CO:COLLECTION_SUMMARY pH7.4). Protein concentrations for both soluble and pellet fractions were CO:COLLECTION_SUMMARY determined by DC protein assay kit (Biorad). For lipidomics of MAMs, the final CO:COLLECTION_SUMMARY banded MAM fraction was washed twice with liquid chromatography-mass CO:COLLECTION_SUMMARY spectrometry (LC-MS) grade PBS and the final MAM pellet was resuspended in LC-MS CO:COLLECTION_SUMMARY grade PBS. Next, 1mL of cold 50% methanol was added and the MAMS were CO:COLLECTION_SUMMARY transferred to glass vials. Chloroform was added (0.5mL) and the mixture was CO:COLLECTION_SUMMARY gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. Lipids were CO:COLLECTION_SUMMARY transferred to a clean glass vial using a glass Hamilton syringe. Lipids were CO:COLLECTION_SUMMARY extracted twice using chloroform prior to being dried under nitrogen gas. CO:COLLECTION_SUMMARY Samples were normalized according to protein concentration. Extracted lipids CO:COLLECTION_SUMMARY were resuspended in a 1:1:1 solution of methanol:chloroform:isopropanol prior to CO:COLLECTION_SUMMARY mass 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. To SP:SAMPLEPREP_SUMMARY assist quantification various volumes were injected for MS (4, 8, and 16ul for SP:SAMPLEPREP_SUMMARY positive mode analysis and 5, 10, and 15ul for negative mode analysis). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Lipids were identified and quantitatively measured using ultra high-performance CH:CHROMATOGRAPHY_SUMMARY liquid-chromatography high-resolution tandem MS/MS (UHPLC-MS/MS) as recently CH:CHROMATOGRAPHY_SUMMARY described89,90. Separation of lipids was done by reverse-phase chromatography CH:CHROMATOGRAPHY_SUMMARY using a Waters ACQUITY CSH C18 column (150x2.1mm; 1.7um) at 60°C using a CH:CHROMATOGRAPHY_SUMMARY Vanquish UHPLC system (Thermo Scientific) and two solvents: solvent A (40:60 CH:CHROMATOGRAPHY_SUMMARY water-methanol plus 10mM ammonium formate and 0.1% formic acid) and solvent B CH:CHROMATOGRAPHY_SUMMARY (10:90 methanol-isopropanol plus 10mM ammonium formate and 0.1% formic acid). CH:CHROMATOGRAPHY_SUMMARY UHPLC was performed at a 0.25 ml per min flow rate for 30 min per sample, CH:CHROMATOGRAPHY_SUMMARY starting at 25% solvent B and ending at 100% solvent B as described. The column CH:CHROMATOGRAPHY_SUMMARY was washed and equilibrated between samples. Samples were run in a semi-random CH:CHROMATOGRAPHY_SUMMARY order where WT or UBXD8 KO samples were interspersed with blank samples. Each CH:CHROMATOGRAPHY_SUMMARY sample was repeatedly analyzed using various injection volumes (4, 8, 16ul for CH:CHROMATOGRAPHY_SUMMARY positive mode and 5, 10, and 15ul for negative mode). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um) 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 El-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 20220508_neg_KO_rep1_10ul 20220508_neg_KO_rep3_10ul 20220508_neg_KO_rep4_10ul 20220508_neg_WT_rep1_10ul 20220508_neg_WT_rep3_10ul 20220508_neg_WT_rep4_10ul 20220820_tufts_MAMfrac_neg_rep8_WT_5ul 20220820_tufts_MAMfrac_neg_rep8_WT_10ul 20220820_tufts_MAMfrac_neg_rep8_WT_15ul 20220820_tufts_MAMfrac_neg_rep8_KO_5ul 20220820_tufts_MAMfrac_neg_rep8_KO_10ul 20220820_tufts_MAMfrac_neg_rep8_KO_15ul Factors Description:UBXD8 Knockout | inj vol:10 Description:UBXD8 Knockout | inj vol:10 Description:UBXD8 Knockout | inj vol:10 Description:Wild-type | inj vol:10 Description:Wild-type | inj vol:10 Description:Wild-type | inj vol:10 Description:Wild-type | inj vol:5 Description:Wild-type | inj vol:10 Description:Wild-type | inj vol:15 Description:UBXD8 Knockout | inj vol:5 Description:UBXD8 Knockout | inj vol:10 Description:UBXD8 Knockout | inj vol:15 PA(32:0) 119372.32 199687.88 290196.5 94546.5 206216.05 251183.69 PA(34:0) 75393.49 143279.61 203150.48 49077 100294.6 136205.31 PA(34:1) 82738.94 131254.52 198519.89 75922.6 132563.69 202664.86 LPE(16:0) 308554.31 121099.69 157617.39 413653.59 154981.95 205565.95 127752.97 179203.11 191873.67 127419.04 144590.23 179717 LPE(18:0) 695039.25 194009.67 341926.34 729851.81 249356.86 404484.34 277510.34 366069.09 408023.5 345247.72 436269.75 521243.41 LPE(18:1) 1384892.38 607755.31 467972.09 1101856.38 1314159.75 528358.06 359493.34 487714.88 557111.75 228876.45 290938.72 343574.97 LPE(20:1) 226720.88 206799.08 168606.2 223223.23 376068.84 149052.2 87086.32 116296.95 121937.34 41741.18 63616.02 80086.36 LPE(22:0) 43708.3 9080.67 13874.02 27428.85 15952.56 11477.48 19064.56 26538.68 26352.9 24614.02 32577.33 41868.59 LPE(22:1) 18580.72 27488.7 30808.07 10241.33 12678.51 15795.27 LPE(22:4) 176836.58 129074.46 157823.52 120398.4 190447.33 77550.44 37159.21 42944.69 59142.8 42959.07 50266.95 58684.31 LPE(22:5) 235823.88 146869.7 169650.06 147548.39 221666.94 82576.59 23437.79 34579.58 38362.46 29803.44 41021.79 44602.66 LPE(24:0) 195752.83 44803.24 41325.34 133196.81 40379.99 34359.55 55717.63 80897.16 97056.7 111553.46 162904.86 191158.08 PE(32:1) 1234533.38 497328.03 553484.94 1710845.62 1251248.88 1242887.38 1043291.06 806036.31 1121851 611463.69 582289.31 618298.94 PE(32:2) 198095.95 237984.39 244271.92 51668.14 63485.89 78782.09 PE(34:1) 8340084 3870593.25 3655652.25 9690195 7428546 6345122.5 5415812 5805066.5 6146950.5 3299313.25 5103298.5 4561514.5 PE(34:2) 1458868.5 529957 546235.06 2219418.75 1690292 1516882.62 1774800.62 2230681.25 2608189.25 621553.06 961922.69 974420.56 PE(34:3) 59878.11 99206 72508.95 22209.36 31619.03 30668.88 PE(36:1) 8016593.5 4251475.5 3177651.75 7648190.5 5905661.5 4299052.5 3743388 4537129.5 4397228.5 3197586 3962134.75 4272067.5 PE(36:2) 7757993.5 3498230 3217456 10645025 7794352 6068283.5 8836895 10423623 10188000 4073630.75 5048322 4407033.5 PE(36:3) 665236.5 945320.69 948338.25 300599.75 437018.16 404995.34 PE(36:4) 486216.75 248164.77 378622.41 591779.06 415015.91 379101.72 298449.88 376714.09 355942.16 259001.3 353962.5 361540.75 PE(36:5) 59076.85 43085.24 34045.4 25661.98 20067.29 17654.43 PE(38:2) 982191.94 514261.59 347781.25 1239520.38 914478.81 590963.81 853834.75 1170742.38 1167199.75 360850.09 509123.62 459703.84 PE(38:4) 2988646 1918954.62 2186340.75 2662352.75 2449436 2054786.12 1516898 1466048.38 1585257 1476582.5 1907863.62 1620858.12 PE(38:5) 1056790.62 361338.25 613335.94 357550.75 223645.48 59397.36 PE(40:1) 448239.84 260336.42 125430.95 602273.06 211052.83 143945.39 157721.36 242035.88 262015.42 134903.48 223206.75 234692.3 PE(40:2) 377614.66 221307.61 131992.69 655127.69 350433.62 207204.8 247054.33 370484.09 382309.16 98781.53 167520.55 147697.2 PE(40:4) 364482 318011.47 332586.09 345528.38 347364.16 262066.61 153903.88 178515.17 146328.08 212507.67 264256.53 269673.47 PE(42:1) 667319.94 339083.66 164585.95 981757.81 290670.22 159706.02 245419.3 378899.53 430610 263299.53 401849.53 479536.78 PE(42:2) 137437.06 117993.27 59744.96 242911.42 118158.79 68746.6 101916.1 142250.77 173713.67 41055.54 67344.98 78377.74 PE(44:1) 103381.13 38811.77 24740.61 103738.87 32718.3 17121.16 22780.89 45488.29 58316.93 35971.57 70113.82 67616.68 PE(44:4) 185374.06 99533.18 57811.53 228590.56 72959.93 33540 37351.51 58549.69 69240.37 51684.68 87926.79 94520.71 PG(32:1) 105799.12 136930.41 152311.67 181474.45 227333.69 270076.59 PG(32:2) 29227.24 37123.29 35109.7 35678.5 44615.69 51075.25 PG(34:1) 429995.28 616993.25 657533.81 673147.62 926755.5 976482.81 PG(34:2) 430970.53 597340.94 603751.81 462300.59 532373.44 636689.94 PG(36:2) 1952736.38 2291027.5 2461043.75 1627811 1952644.5 2267087.75 PG(36:3) 155898.39 195455.77 194711.31 96296.62 134577.94 150875.38 PG(38:3) 52322.24 69309.6 73020.95 32058.55 43697.62 47600.61 PG(40:7) 160357.44 190261.95 218499.42 214939.88 245258.81 302136.66 PI(32:0) 209028.8 260553.86 286840.47 368652.53 438176.84 531693.94 PI(34:0) 266797.12 351087.72 296398.47 452364 482161.34 429456.38 PI(34:1) 2616655 709296.81 1437600.12 2851011.75 948202.81 1406428.5 3075107.75 3786797.25 4273603.5 3530307.25 4004018.75 5002317 PI(34:2) 773326.25 1046133.19 1048403.81 427843.25 543069.31 656661.19 PI(36:1) 5045960.5 913366.75 1698506 5007920.5 1318022.62 1636848.5 5309750.5 4155685.25 5139957.5 7676557.5 7565681.5 7643934.5 PI(36:2) 1935115.62 558472.25 1054139 2655405.75 1141293.12 1397949.5 3396432.75 4358778 4486292.5 2282344.75 2764725 3002488.75 PI(36:3) 351449.03 488259 518149 226796.62 307126.53 382885.75 PI(36:4) 593350.19 765454.94 813960.06 447590.97 610462.12 688226.06 PI(38:1) 511999.34 130464.88 180235.55 301857.16 117134.65 147806.58 424951.16 402066.78 496285.5 796061.44 879136.75 765543.25 PI(38:2) 804216.75 148380.02 324160.25 1116022.5 398223.41 367664.12 896537.69 1050126.5 1326964 677833.31 1011637.75 1063998.62 PI(38:3) 1304018.88 331201.78 740142.44 1612740.88 624734.56 732057.5 1676476.88 2289680.75 1692118.88 1528113.38 1813301 1680214 PI(38:4) 3463428.75 967732.69 2376483.25 4190495 2366345.75 2311957 5895250 7017581.5 6846464.5 5430855 5650369.5 6008806 PI(38:5) 464240.09 161364.39 373988.75 591864.94 332112.03 451666.84 1207728.62 1513318.12 1629019.38 665630.5 819823.31 1012753.69 PI(40:4) 163155.61 54734.59 133770.67 254209.83 69145.45 93653.04 305680.22 251028.44 313220.97 392141.78 370736.84 390244.34 PI(40:5) 247248.5 277760.91 287978.41 253706.8 332255.59 322739.47 PI(40:6) 202228.14 53453.34 110021.96 153496.61 103915.56 112214.62 314991.66 379467.03 360573.28 332621.03 370734.5 358160.16 LPS(18:0) 374735.66 131722.69 111859.96 319891.72 109534.48 108345.48 107973.34 181796.45 245526.73 141418.25 211667.33 297696.09 LPS(20:0) 5552.13 10351.63 10638 7111.61 10281.42 12399.31 LPS(24:0) 14671.52 28743.71 43791.73 45898.96 78628.12 123916.52 PS(32:1) 394175.62 245260.67 255765.55 528305.06 289952.53 260564.7 166110.05 271391.91 364729.41 119036.4 205855.45 264748.66 PS(34:1) 3368704.25 1821886 1849955.62 4852153.5 2389062 1877015 1404132 2385982.25 2574428.25 1094951 1638071.62 2018192.62 PS(34:2) 207602.17 92421.79 88114.74 387073.03 177797.2 139493.61 189922.61 303773.91 388515.47 83819.38 131888.14 197876.11 PS(36:1) 4013800.25 2053760 2288342 5008565.5 2606342.25 2067728.5 1884814.62 2783927.75 3763746.75 1357592.62 2433823.25 2892370.75 PS(36:2) 1659768.12 651450.69 751427.94 2520577.25 1279670.88 790714.69 1144759.12 1760778.62 1843309.38 547758.31 875038.31 835866.31 PS(38:1) 662589.69 350442.5 273029.38 653546.19 291698.47 181922.7 PS(38:2) 475528.84 227698.88 195353.12 798173.44 381374.78 240987.98 PS(38:3) 125339.73 143381.81 175380.95 205808.2 163080.44 124836.31 PS(38:4) 264252.5 115535.88 160065.5 276262.56 155377.77 126651.16 PS(40:1) 1029986.75 545423.81 302708.78 884198.31 394183.59 204003.77 PS(40:2) 479429.91 271835.5 195016.89 688143.62 406196.25 230529.19 PS(40:4) 178762.75 182350.88 288602.81 234680 167679.41 143284.78 PS(40:5) 257245.42 192242.38 327701.25 274262.84 193055.05 183405.17 PS(40:6) 271043.84 198898.17 297821.16 221978 247436.98 228293.7 PS(42:1) 906004.25 466784.72 150279.97 822522.25 261493.61 98862.23 PS(42:2) 140681.89 137274.81 67198.36 174734.06 102583.57 63802.67 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 20220508_neg_KO_rep1_10ul 20220508_neg_KO_rep3_10ul 20220508_neg_KO_rep4_10ul 20220508_neg_WT_rep1_10ul 20220508_neg_WT_rep3_10ul 20220508_neg_WT_rep4_10ul 20220820_tufts_MAMfrac_neg_rep8_WT_5ul 20220820_tufts_MAMfrac_neg_rep8_WT_10ul 20220820_tufts_MAMfrac_neg_rep8_WT_15ul 20220820_tufts_MAMfrac_neg_rep8_KO_5ul 20220820_tufts_MAMfrac_neg_rep8_KO_10ul 20220820_tufts_MAMfrac_neg_rep8_KO_15ul PA(32:0) 119372.32 199687.88 290196.5 94546.5 206216.05 251183.69 PA(34:0) 75393.49 143279.61 203150.48 49077 100294.6 136205.31 PA(34:1) 82738.94 131254.52 198519.89 75922.6 132563.69 202664.86 LPE(16:0) 308554.31 121099.69 157617.39 413653.59 154981.95 205565.95 127752.97 179203.11 191873.67 127419.04 144590.23 179717 LPE(18:0) 695039.25 194009.67 341926.34 729851.81 249356.86 404484.34 277510.34 366069.09 408023.5 345247.72 436269.75 521243.41 LPE(18:1) 1384892.38 607755.31 467972.09 1101856.38 1314159.75 528358.06 359493.34 487714.88 557111.75 228876.45 290938.72 343574.97 LPE(20:1) 226720.88 206799.08 168606.2 223223.23 376068.84 149052.2 87086.32 116296.95 121937.34 41741.18 63616.02 80086.36 LPE(22:0) 43708.3 9080.67 13874.02 27428.85 15952.56 11477.48 19064.56 26538.68 26352.9 24614.02 32577.33 41868.59 LPE(22:1) 18580.72 27488.7 30808.07 10241.33 12678.51 15795.27 LPE(22:4) 176836.58 129074.46 157823.52 120398.4 190447.33 77550.44 37159.21 42944.69 59142.8 42959.07 50266.95 58684.31 LPE(22:5) 235823.88 146869.7 169650.06 147548.39 221666.94 82576.59 23437.79 34579.58 38362.46 29803.44 41021.79 44602.66 LPE(24:0) 195752.83 44803.24 41325.34 133196.81 40379.99 34359.55 55717.63 80897.16 97056.7 111553.46 162904.86 191158.08 PE(32:1) 1234533.38 497328.03 553484.94 1710845.62 1251248.88 1242887.38 1043291.06 806036.31 1121851 611463.69 582289.31 618298.94 PE(32:2) 198095.95 237984.39 244271.92 51668.14 63485.89 78782.09 PE(34:1) 8340084 3870593.25 3655652.25 9690195 7428546 6345122.5 5415812 5805066.5 6146950.5 3299313.25 5103298.5 4561514.5 PE(34:2) 1458868.5 529957 546235.06 2219418.75 1690292 1516882.62 1774800.62 2230681.25 2608189.25 621553.06 961922.69 974420.56 PE(34:3) 59878.11 99206 72508.95 22209.36 31619.03 30668.88 PE(36:1) 8016593.5 4251475.5 3177651.75 7648190.5 5905661.5 4299052.5 3743388 4537129.5 4397228.5 3197586 3962134.75 4272067.5 PE(36:2) 7757993.5 3498230 3217456 10645025 7794352 6068283.5 8836895 10423623 10188000 4073630.75 5048322 4407033.5 PE(36:3) 665236.5 945320.69 948338.25 300599.75 437018.16 404995.34 PE(36:4) 486216.75 248164.77 378622.41 591779.06 415015.91 379101.72 298449.88 376714.09 355942.16 259001.3 353962.5 361540.75 PE(36:5) 59076.85 43085.24 34045.4 25661.98 20067.29 17654.43 PE(38:2) 982191.94 514261.59 347781.25 1239520.38 914478.81 590963.81 853834.75 1170742.38 1167199.75 360850.09 509123.62 459703.84 PE(38:4) 2988646 1918954.62 2186340.75 2662352.75 2449436 2054786.12 1516898 1466048.38 1585257 1476582.5 1907863.62 1620858.12 PE(38:5) 1056790.62 361338.25 613335.94 357550.75 223645.48 59397.36 PE(40:1) 448239.84 260336.42 125430.95 602273.06 211052.83 143945.39 157721.36 242035.88 262015.42 134903.48 223206.75 234692.3 PE(40:2) 377614.66 221307.61 131992.69 655127.69 350433.62 207204.8 247054.33 370484.09 382309.16 98781.53 167520.55 147697.2 PE(40:4) 364482 318011.47 332586.09 345528.38 347364.16 262066.61 153903.88 178515.17 146328.08 212507.67 264256.53 269673.47 PE(42:1) 667319.94 339083.66 164585.95 981757.81 290670.22 159706.02 245419.3 378899.53 430610 263299.53 401849.53 479536.78 PE(42:2) 137437.06 117993.27 59744.96 242911.42 118158.79 68746.6 101916.1 142250.77 173713.67 41055.54 67344.98 78377.74 PE(44:1) 103381.13 38811.77 24740.61 103738.87 32718.3 17121.16 22780.89 45488.29 58316.93 35971.57 70113.82 67616.68 PE(44:4) 185374.06 99533.18 57811.53 228590.56 72959.93 33540 37351.51 58549.69 69240.37 51684.68 87926.79 94520.71 PG(32:1) 105799.12 136930.41 152311.67 181474.45 227333.69 270076.59 PG(32:2) 29227.24 37123.29 35109.7 35678.5 44615.69 51075.25 PG(34:1) 429995.28 616993.25 657533.81 673147.62 926755.5 976482.81 PG(34:2) 430970.53 597340.94 603751.81 462300.59 532373.44 636689.94 PG(36:2) 1952736.38 2291027.5 2461043.75 1627811 1952644.5 2267087.75 PG(36:3) 155898.39 195455.77 194711.31 96296.62 134577.94 150875.38 PG(38:3) 52322.24 69309.6 73020.95 32058.55 43697.62 47600.61 PG(40:7) 160357.44 190261.95 218499.42 214939.88 245258.81 302136.66 PI(32:0) 209028.8 260553.86 286840.47 368652.53 438176.84 531693.94 PI(34:0) 266797.12 351087.72 296398.47 452364 482161.34 429456.38 PI(34:1) 2616655 709296.81 1437600.12 2851011.75 948202.81 1406428.5 3075107.75 3786797.25 4273603.5 3530307.25 4004018.75 5002317 PI(34:2) 773326.25 1046133.19 1048403.81 427843.25 543069.31 656661.19 PI(36:1) 5045960.5 913366.75 1698506 5007920.5 1318022.62 1636848.5 5309750.5 4155685.25 5139957.5 7676557.5 7565681.5 7643934.5 PI(36:2) 1935115.62 558472.25 1054139 2655405.75 1141293.12 1397949.5 3396432.75 4358778 4486292.5 2282344.75 2764725 3002488.75 PI(36:3) 351449.03 488259 518149 226796.62 307126.53 382885.75 PI(36:4) 593350.19 765454.94 813960.06 447590.97 610462.12 688226.06 PI(38:1) 511999.34 130464.88 180235.55 301857.16 117134.65 147806.58 424951.16 402066.78 496285.5 796061.44 879136.75 765543.25 PI(38:2) 804216.75 148380.02 324160.25 1116022.5 398223.41 367664.12 896537.69 1050126.5 1326964 677833.31 1011637.75 1063998.62 PI(38:3) 1304018.88 331201.78 740142.44 1612740.88 624734.56 732057.5 1676476.88 2289680.75 1692118.88 1528113.38 1813301 1680214 PI(38:4) 3463428.75 967732.69 2376483.25 4190495 2366345.75 2311957 5895250 7017581.5 6846464.5 5430855 5650369.5 6008806 PI(38:5) 464240.09 161364.39 373988.75 591864.94 332112.03 451666.84 1207728.62 1513318.12 1629019.38 665630.5 819823.31 1012753.69 PI(40:4) 163155.61 54734.59 133770.67 254209.83 69145.45 93653.04 305680.22 251028.44 313220.97 392141.78 370736.84 390244.34 PI(40:5) 247248.5 277760.91 287978.41 253706.8 332255.59 322739.47 PI(40:6) 202228.14 53453.34 110021.96 153496.61 103915.56 112214.62 314991.66 379467.03 360573.28 332621.03 370734.5 358160.16 LPS(18:0) 374735.66 131722.69 111859.96 319891.72 109534.48 108345.48 107973.34 181796.45 245526.73 141418.25 211667.33 297696.09 LPS(20:0) 5552.13 10351.63 10638 7111.61 10281.42 12399.31 LPS(24:0) 14671.52 28743.71 43791.73 45898.96 78628.12 123916.52 PS(32:1) 394175.62 245260.67 255765.55 528305.06 289952.53 260564.7 166110.05 271391.91 364729.41 119036.4 205855.45 264748.66 PS(34:1) 3368704.25 1821886 1849955.62 4852153.5 2389062 1877015 1404132 2385982.25 2574428.25 1094951 1638071.62 2018192.62 PS(34:2) 207602.17 92421.79 88114.74 387073.03 177797.2 139493.61 189922.61 303773.91 388515.47 83819.38 131888.14 197876.11 PS(36:1) 4013800.25 2053760 2288342 5008565.5 2606342.25 2067728.5 1884814.62 2783927.75 3763746.75 1357592.62 2433823.25 2892370.75 PS(36:2) 1659768.12 651450.69 751427.94 2520577.25 1279670.88 790714.69 1144759.12 1760778.62 1843309.38 547758.31 875038.31 835866.31 PS(38:1) 662589.69 350442.5 273029.38 653546.19 291698.47 181922.7 PS(38:2) 475528.84 227698.88 195353.12 798173.44 381374.78 240987.98 PS(38:3) 125339.73 143381.81 175380.95 205808.2 163080.44 124836.31 PS(38:4) 264252.5 115535.88 160065.5 276262.56 155377.77 126651.16 PS(40:1) 1029986.75 545423.81 302708.78 884198.31 394183.59 204003.77 PS(40:2) 479429.91 271835.5 195016.89 688143.62 406196.25 230529.19 PS(40:4) 178762.75 182350.88 288602.81 234680 167679.41 143284.78 PS(40:5) 257245.42 192242.38 327701.25 274262.84 193055.05 183405.17 PS(40:6) 271043.84 198898.17 297821.16 221978 247436.98 228293.7 PS(42:1) 906004.25 466784.72 150279.97 822522.25 261493.61 98862.23 PS(42:2) 140681.89 137274.81 67198.36 174734.06 102583.57 63802.67 METABOLITES_END #END