#METABOLOMICS WORKBENCH jad2033_20230615_184322 DATATRACK_ID:4093 STUDY_ID:ST002745 ANALYSIS_ID:AN004451 PROJECT_ID:PR001509 VERSION 1 CREATED_ON June 26, 2023, 11:30 am #PROJECT PR:PROJECT_TITLE Biomolecular condensates create phospholipid-enriched microenvironments PR:PROJECT_TYPE Metabolomics of in vitro condensates PR:PROJECT_SUMMARY Proteins and RNA are able to phase separate from the aqueous cellular PR:PROJECT_SUMMARY environment to form sub-cellular compartments called condensates. This process PR:PROJECT_SUMMARY results in a protein-RNA mixture that is chemically distinct from the PR:PROJECT_SUMMARY surrounding aqueous phase. Here we use mass spectrometry to characterize the PR:PROJECT_SUMMARY metabolomes of condensates. To test this, we prepared mixtures of PR:PROJECT_SUMMARY phase-separated proteins and cellular metabolites and identified metabolites PR:PROJECT_SUMMARY enriched in the condensate phase. These proteins included SARS-CoV-2 PR:PROJECT_SUMMARY nucleocapsid, as well as low complexity domains of MED1 and HNRNPA1. PR:INSTITUTE Cornell University PR:DEPARTMENT Department of Pharmacology PR:LABORATORY Dr. Samie Jaffrey PR:LAST_NAME Dumelie PR:FIRST_NAME Jason PR:ADDRESS 1300 York Ave, LC-524, New York City, NY PR:EMAIL srj2003@med.cornell.edu PR:PHONE 6465690174 PR:FUNDING_SOURCE This work was supported by the National Institutes of Health grants R35NS111631 PR:FUNDING_SOURCE and R01CA186702 (S.R.J.); R01AR076029, R21ES032347 and R21NS118633 (Q.C.); and PR:FUNDING_SOURCE NIH P01 HD067244 and support from the Starr Cancer Consortium I13-0037 (S.S.G.). PR:PUBLICATIONS Under revision PR:CONTRIBUTORS Jason G. Dumelie, Qiuying Chen, Dawson Miller, Nabeel Attarwala, Steven S. Gross PR:CONTRIBUTORS and Samie R. Jaffrey1 #STUDY ST:STUDY_TITLE Biomolecular condensates create phospholipid-enriched microenvironments (Part 6) ST:STUDY_SUMMARY Proteins and RNA are able to phase separate from the aqueous cellular ST:STUDY_SUMMARY environment to form sub-cellular compartments called condensates. This process ST:STUDY_SUMMARY results in a protein-RNA mixture that is chemically distinct from the ST:STUDY_SUMMARY surrounding aqueous phase. Here we use mass spectrometry to characterize the ST:STUDY_SUMMARY metabolomes of condensates. To test this, we prepared mixtures of ST:STUDY_SUMMARY phase-separated proteins and cellular metabolites and identified metabolites ST:STUDY_SUMMARY enriched in the condensate phase. Here, we quantified the concentration of a ST:STUDY_SUMMARY select set of phospholipids in the aqueous and condensate phase of condensates ST:STUDY_SUMMARY formed from the low complexity domain of MED1 by comparison with ST:STUDY_SUMMARY isotopic-labeled phospholipid standards. ST:INSTITUTE Cornell University ST:DEPARTMENT Department of Pharmacology ST:LABORATORY Dr. Samie Jaffrey ST:LAST_NAME Dumelie ST:FIRST_NAME Jason ST:ADDRESS 1300 York Ave, LC-524, New York City, NY ST:EMAIL srj2003@med.cornell.edu ST:STUDY_TYPE Metabolomes of in vitro synthesized condensates ST:PHONE 6465690174 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - MED1 PE Aqueous Sample 5 fraction:aqueous | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=5; RAW_FILE_NAME=PE52T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PE Aqueous Sample 6 fraction:aqueous | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=5; RAW_FILE_NAME=PE53T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PE Aqueous Sample 9 fraction:aqueous | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=5; RAW_FILE_NAME=PE63T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PE Condensate Sample 5 fraction:condensate | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=5; RAW_FILE_NAME=PE52B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PE Condensate Sample 6 fraction:condensate | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=5; RAW_FILE_NAME=PE53B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PE Condensate Sample 9 fraction:condensate | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=5; RAW_FILE_NAME=PE63B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PE Input Sample 5 fraction:input | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=5; RAW_FILE_NAME=PE52I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PE Input Sample 6 fraction:input | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=5; RAW_FILE_NAME=PE53I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PE Input Sample 9 fraction:input | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=5; RAW_FILE_NAME=PE63I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PI Aqueous Sample 5 fraction:aqueous | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=3; RAW_FILE_NAME=PI52T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PI Aqueous Sample 6 fraction:aqueous | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=3; RAW_FILE_NAME=PI53T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PI Aqueous Sample 9 fraction:aqueous | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=3; RAW_FILE_NAME=PI63T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PI Condensate Sample 5 fraction:condensate | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=3; RAW_FILE_NAME=PI52B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PI Condensate Sample 6 fraction:condensate | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=3; RAW_FILE_NAME=PI53B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PI Condensate Sample 9 fraction:condensate | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=3; RAW_FILE_NAME=PI63B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PI Input Sample 5 fraction:input | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=3; RAW_FILE_NAME=PI52I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PI Input Sample 6 fraction:input | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 20; Sample injection volume (µL)=3; RAW_FILE_NAME=PI53I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PI Input Sample 9 fraction:input | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 300; Sample injection volume (µL)=3; RAW_FILE_NAME=PI63I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PO Aqueous Sample 5 fraction:aqueous | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 300; Sample injection volume (µL)=5; RAW_FILE_NAME=PO52T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PO Aqueous Sample 6 fraction:aqueous | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 300; Sample injection volume (µL)=5; RAW_FILE_NAME=PO53T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PO Aqueous Sample 9 fraction:aqueous | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 300; Sample injection volume (µL)=5; RAW_FILE_NAME=PO63T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PO Condensate Sample 5 fraction:condensate | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 300; Sample injection volume (µL)=5; RAW_FILE_NAME=PO52B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PO Condensate Sample 6 fraction:condensate | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 300; Sample injection volume (µL)=5; RAW_FILE_NAME=PO53B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PO Condensate Sample 9 fraction:condensate | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 300; Sample injection volume (µL)=5; RAW_FILE_NAME=PO63B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PO Input Sample 5 fraction:input | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 300; Sample injection volume (µL)=5; RAW_FILE_NAME=PO52I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PO Input Sample 6 fraction:input | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 300; Sample injection volume (µL)=5; RAW_FILE_NAME=PO53I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 PO Input Sample 9 fraction:input | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; SPLASH™ LIPIDOMIX® Quantitative Mass Spec Internal Standard dilution=1 in 300; Sample injection volume (µL)=5; RAW_FILE_NAME=PO63I.mzdata.xml #COLLECTION CO:COLLECTION_SUMMARY Mouse metabolites were collected from the liver of female mice using methanol CO:COLLECTION_SUMMARY extraction. After euthanizing a mouse, the liver was immediately frozen in CO:COLLECTION_SUMMARY liquid nitrogen. We then used cold 80% methanol to extract metabolites. First, 1 CO:COLLECTION_SUMMARY ml of 80% methanol was added to the liver and incubated for 10 min at -20oC. CO:COLLECTION_SUMMARY Glass beads were added to the liver and then the liver was lysed by bead-beating CO:COLLECTION_SUMMARY for 45 s using a Tissuelyser cell disrupter (Qiagen). The lysate was incubated CO:COLLECTION_SUMMARY for 10 min at -20oC and centrifuged (13200 rpm, 5 min) to separate metabolites CO:COLLECTION_SUMMARY from macromolecules. The supernatant was collected and 200 µl of 80% methanol CO:COLLECTION_SUMMARY was added to the pellet. The incubation, shaking and centrifugation steps were CO:COLLECTION_SUMMARY repeated twice to extract more metabolites from the pellet. The three CO:COLLECTION_SUMMARY supernatants were combined and centrifuged (14000 rpm, 10 min) to separate any CO:COLLECTION_SUMMARY remaining macromolecules from the metabolites. The combined supernatants were CO:COLLECTION_SUMMARY dried using a SpeedVac Concentrator (Savant, SPD131DDA) at 25oC and the dried CO:COLLECTION_SUMMARY metabolite samples were stored at -80oC. The amount of protein in the pellet was CO:COLLECTION_SUMMARY measured using the Quick Start Bradford assay to calculate the metabolites’ CO:COLLECTION_SUMMARY protein equivalent mass. Mouse metabolites were initially re-suspended in CO:COLLECTION_SUMMARY condensate buffer (50 mM NH4HCO3 pH 7.5, 50 mM NaCl, 1 mM DTT) to a protein CO:COLLECTION_SUMMARY equivalent concentration of 938 g/l. The chosen final concentration of CO:COLLECTION_SUMMARY metabolites is slightly lower than the 200-300 g/l protein concentration CO:COLLECTION_SUMMARY observed in cells. Metabolites that were not fully soluble in condensate buffer CO:COLLECTION_SUMMARY were removed by centrifugation (2x5 min, 16,000 g each), in which only the CO:COLLECTION_SUMMARY supernatant was retained. Purified mCherry tagged MED1 low-complexity domain CO:COLLECTION_SUMMARY (37.5 μM) was centrifuged (1 min, 1,000 g) to disrupt any existing condensates CO:COLLECTION_SUMMARY and to remove any precipitated proteins. The MED1 (final concentration, 30 μM) CO:COLLECTION_SUMMARY was then combined with metabolites (final concentration, 150 g/l protein CO:COLLECTION_SUMMARY equivalent) and then phage lambda RNA (final concentration, 0.15 μM) in a total CO:COLLECTION_SUMMARY volume of 300 µl. An input sample (10 µl) was saved and then the sample was CO:COLLECTION_SUMMARY allowed to incubate for 10 min at 25oC. Condensates were then separated from the CO:COLLECTION_SUMMARY aqueous environment by centrifugation (10 min, 12,500 g, 25oC). The aqueous CO:COLLECTION_SUMMARY phase was removed from the condensate phase and then equal volumes (usually ~ 2 CO:COLLECTION_SUMMARY µl) of the aqueous fraction, condensate fraction and input sample were CO:COLLECTION_SUMMARY processed for metabolomics using identical approaches as described below. First CO:COLLECTION_SUMMARY the samples were diluted in ammonium bicarbonate buffer (50 mM NH4HCO3 pH 7.5) CO:COLLECTION_SUMMARY and briefly heated (2 min, 65oC) to disrupt condensates before being added CO:COLLECTION_SUMMARY immediately to 4x volume of ice-cold 100% methanol to precipitate protein and CO:COLLECTION_SUMMARY RNA. This heating step was excluded for some samples where noted. Protein and CO:COLLECTION_SUMMARY RNA were separated from metabolites by vortexing the samples (2 min), followed CO:COLLECTION_SUMMARY by incubation at -25oC (10 min) and then centrifugation (5 min, 13,000 rpm). The CO:COLLECTION_SUMMARY supernatant was saved and the process was repeated on the pellet two more times CO:COLLECTION_SUMMARY after adding 200 µl of 80% methanol each time to the pellet. The three CO:COLLECTION_SUMMARY supernatants were combined and centrifuged (10 min, 14000 rpm) to remove any CO:COLLECTION_SUMMARY additional macromolecules. The final supernatant was collected and dried using a CO:COLLECTION_SUMMARY SpeedVac Concentrator run at 25oC. Notably, in one subset of experiments, CO:COLLECTION_SUMMARY metabolites were added to MED1 condensates after the 10 min incubation rather CO:COLLECTION_SUMMARY than prior to the incubation. CO:SAMPLE_TYPE Liver CO:COLLECTION_METHOD 80% methanol CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Mouse liver metabolites were combined with the condensate-forming low-complexity TR:TREATMENT_SUMMARY domain of MED1. Condensates were stimulated with 150 nM RNA and then incubated TR:TREATMENT_SUMMARY for 10 min. In a subset of samples, RNA addition occurred 10 min before TR:TREATMENT_SUMMARY metabolite addition. Next, condensates were centrifuged to the bottom of a 600 TR:TREATMENT_SUMMARY ul tube. Equal fractions from the input sample, aqueous phase and condensate TR:TREATMENT_SUMMARY phases were collected separately. Metabolites were extracted from each fraction TR:TREATMENT_SUMMARY using 80% methanol in steps that involved disrupting condensates with heat. In a TR:TREATMENT_SUMMARY subset of samples, this heat step was omitted. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Dried-down extracts were reconstituted in 150 µl 70% acetonitrile, at a SP:SAMPLEPREP_SUMMARY relative protein concentration of ~ 2 µg/µl. These were stored until used at SP:SAMPLEPREP_SUMMARY -20C. SPLASH® LIPIDOMIX® Quantitative Mass Spec Standard was diluted 1/30 or SP:SAMPLEPREP_SUMMARY 1/200, combined with the reconstituted extract and then either 3 µl (for PIs SP:SAMPLEPREP_SUMMARY samples) or 5 µl (all other samples) were injected for LC/MS-based targeted SP:SAMPLEPREP_SUMMARY metabolite profiling. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -20℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatography of metabolites utilized reversed phase chromatography on a CH:CHROMATOGRAPHY_SUMMARY Agilent ZORBAX Eclipse Plus C18, 100 × 2.1 mm, 1.8 μm. Mobile phases consisted CH:CHROMATOGRAPHY_SUMMARY of (A) 10 mM ammonium formate with 5 μM Agilent deactivator additive in 5:3:2 CH:CHROMATOGRAPHY_SUMMARY water:acetonitrile:2-propanol and (B) 10 mM ammonium formate in 1:9:90 CH:CHROMATOGRAPHY_SUMMARY water:acetonitrile:2-propanol. Column temperature was set at 60°C and CH:CHROMATOGRAPHY_SUMMARY autosampler temperature was at 20°C. The flow rate was 0.4 mL/min. The CH:CHROMATOGRAPHY_SUMMARY following gradient was applied: 0 min, 15% B; 0-2.5 min, to 50% B; 2.5-2.6 min, CH:CHROMATOGRAPHY_SUMMARY to 57%, 2.6-9 min, to 70% B; 9-9.1 min, to 93% B; 9.1-11.1 min, to 96%; 11.1- CH:CHROMATOGRAPHY_SUMMARY 15min, 100% B; 15-20 min, 15% B. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent Model 1290 Infinity II liquid chromatography system CH:COLUMN_NAME Cogent Diamond Hydride (150 × 2.1 mm, 4um) CH:SOLVENT_A 50% water/30% acetonitrile/20% isopropanol;10 mM ammonium formate with 5 µM CH:SOLVENT_A Agilent deactivator additive CH:SOLVENT_B 1% water/9% acetonitrile/90% isopropanol;10 mM ammonium formate CH:FLOW_GRADIENT 0 min, 15% B; 0-2.5 min, to 50% B; 2.5-2.6 min, to 57%, 2.6-9 min, to 70% B; CH:FLOW_GRADIENT 9-9.1 min, to 93% B; 9.1-11.1 min, to 96%; 11.1- 15min, 100% B; 15-20 min, 15% CH:FLOW_GRADIENT B. CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6460 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE Other MS:ION_MODE POSITIVE MS:MS_COMMENTS (PE, PC, SM, others)LC/MS-based targeted metabolite profiling. MS mode was a MS:MS_COMMENTS Agilent Jet Stream ESI. To quantify phospholipid concentrations, we used stable MS:MS_COMMENTS isotope standards and dynamic multiple reaction monitoring (dMRM). The MS:MS_COMMENTS transitions for lipid standards were monitored as recommended by the supplier. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Ion abundance (peak area) MS_METABOLITE_DATA_START Samples MED1 PE Aqueous Sample 5 MED1 PE Input Sample 5 MED1 PE Condensate Sample 5 MED1 PE Aqueous Sample 6 MED1 PE Input Sample 6 MED1 PE Condensate Sample 6 MED1 PE Aqueous Sample 9 MED1 PE Input Sample 9 MED1 PE Condensate Sample 9 MED1 PO Aqueous Sample 5 MED1 PO Input Sample 5 MED1 PO Condensate Sample 5 MED1 PO Aqueous Sample 6 MED1 PO Input Sample 6 MED1 PO Condensate Sample 6 MED1 PO Aqueous Sample 9 MED1 PO Input Sample 9 MED1 PO Condensate Sample 9 Factors fraction:aqueous | Extraction from condensate:No heat step fraction:input | Extraction from condensate:No heat step fraction:condensate | Extraction from condensate:No heat step fraction:aqueous | Extraction from condensate:No heat step fraction:input | Extraction from condensate:No heat step fraction:condensate | Extraction from condensate:No heat step fraction:aqueous | Extraction from condensate:standard fraction:input | Extraction from condensate:standard fraction:condensate | Extraction from condensate:standard fraction:aqueous | Extraction from condensate:No heat step fraction:input | Extraction from condensate:No heat step fraction:condensate | Extraction from condensate:No heat step fraction:aqueous | Extraction from condensate:No heat step fraction:input | Extraction from condensate:No heat step fraction:condensate | Extraction from condensate:No heat step fraction:aqueous | Extraction from condensate:standard fraction:input | Extraction from condensate:standard fraction:condensate | Extraction from condensate:standard PE 38:6 60986 60059 530673 40346 57652 468146 35325 47990 618172 PE 34:1 4262 13457 541547 3820 6624 275373 2176 5553 969403 PE 34:2 12936 19429 485547 10013 17388 266574 8042 12389 799320 PE 36:4 19314 22274 181853 13890 18935 155867 14757 20265 209721 PE 40:7 16009 15641 136503 11811 18927 122368 11286 15020 152665 PE 38:4 18514 20394 142427 12587 18564 119890 11117 17110 162066 PE 38:5 34815 34724 279140 19874 35734 239997 20797 28889 318955 PE 36:3 12019 11600 97498 8675 11069 83403 8458 11903 105484 PE 15:0/18:1-d7 4847 5576 5390 5441 5727 4339 4431 4470 4771 LPC 16:1 1639 3037 35372 35 1033 12913 45 4 20509 LPC 18:2 24638 37677 498290 2073 11208 176471 1302 1419 291353 LPC 16:0 90765 124673 1561809 7854 40027 542749 4066 4900 974311 LPC 18:0 33870 48332 548461 2371 15964 184233 1358 1771 349172 LPC 18:1-d7 884565 1018333 865493 73069 365812 377110 29852 36808 572880 PS 38:4 291 382 3515 12 175 1100 9 11 1700 PS 15:0/18:1-d7 21937 24135 19200 1313 7833 10213 243 485 9439 SM d34:1 10372 12689 102208 1035 3773 26808 907 33 50549 SM d18:1/18:1-d9 1359047 1486925 1236489 102492 443737 453922 35183 40020 597605 PC 36:4 39806 49365 410204 5683 21895 144308 238 866 203778 PC 15:0/18:1-d7 2741227 3104350 2528619 219405 1039575 976186 86546 90532 1356322 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Formula Mass (actual) Monitored Transition Retention time (min) PE 38:6 C43H74NO8P 763.5152 (764.5-->623.5) 4.725 PE 34:1 C39H76NO8P 717.5309 (718.5-->577.5) 5.046 PE 34:2 C39H74NO8P 715.5152 (716.5-->575.5) 4.745 PE 36:4 C41H74NO8P 739.5152 (740.5-->599.5) 4.76 PE 40:7 C45H76NO8P 789.5309 (790.5-->649.5) 4.782 PE 38:4 C43H78NO8P 767.5465 (768.6-->627.5) 5.202 PE 38:5 C43H76NO8P 765.5309 (766.6-->625.5) 4.8 PE 36:3 C41H76NO8P 741.530857 (742.5-->601.5) 4.81 PE 15:0/18:1-d7 C38H67D7NO8P 710.559 (711.5-->346.3) 4.846 LPC 16:1 C24H48NO7P 493.3168 (494.3-->184.1) 2.152 LPC 18:2 C26H50NO7P 519.3325 (520.3-->184.1) 2.26 LPC 16:0 C24H50NO7P 495.3325 (496.3-->184.1) 2.47 LPC 18:0 C26H54NO7P 523.3638 (524.4-->184.1) 2.963 LPC 18:1-d7 C26H45D7NO7P 528.392 (529.4-->184.1) 2.56 PS 38:4 C44H78NO10P 811.5363 (812.5-->627.5) 4.08 PS 15:0/18:1-d7 C39H66D7NNaO10P 776.531 (755.5-->570.6) 3.889 SM d34:1 C39H80N2O6P 703.5754 (703.6-->184.1) 4.32 SM d18:1/18:1-d9 C41H72D9N2O6P 737.64 (738.6-->184.1) 4.36 PC 36:4 C44H80NO8P 781.5622 (782.6-->184.1) 5.262 PC 15:0/18:1-d7 C41H73D7NO8P (753.6-->184.1) 5.498 METABOLITES_END #END