#METABOLOMICS WORKBENCH jad2033_20230613_095235 DATATRACK_ID:4089 STUDY_ID:ST002751 ANALYSIS_ID:AN004463 VERSION 1 CREATED_ON 02-08-2024 #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 jdumes98@gmail.com 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:DOI http://dx.doi.org/10.21228/M8N71K 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 5) ST:STUDY_TYPE Metabolomes of mouse liver ST:STUDY_SUMMARY In this study we used LC-MS and MS/MS to characterize the metabolomes of the ST:STUDY_SUMMARY input mouse liver metabolites used in the first two studies of this submission. 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:PHONE 6465690174 ST:SUBMIT_DATE 2023-06-14 #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]Additional sample data SUBJECT_SAMPLE_FACTORS - ANP_experiment_set_3_replicate_2 Factor:2 μl RAW_FILE_NAME=jason liver anpneg1.mzdata.xml jason liver anppos1.mzdata.xml SUBJECT_SAMPLE_FACTORS - ANP_experiment_set_3_replicate_3 Factor:2 μl RAW_FILE_NAME=jason liver anpneg1 2.mzdata.xml jason liver anppos1 2.mzdata.xml SUBJECT_SAMPLE_FACTORS - ANP_experiment_set_3_replicate_4 Factor:2 μl RAW_FILE_NAME=jason liver anpneg1 3.mzdata.xml jason liver anppos1 3.mzdata.xml SUBJECT_SAMPLE_FACTORS - ANP_experiment_set_3_replicate_5 Factor:2 μl RAW_FILE_NAME=jason liver anpneg1 4.mzdata.xml jason liver anppos1 4.mzdata.xml SUBJECT_SAMPLE_FACTORS - ANP_experiment_set_3_replicate_6 Factor:2 μl RAW_FILE_NAME=jason liver anpneg2.mzdata.xml jason liver anppos2.mzdata.xml SUBJECT_SAMPLE_FACTORS - ANP_experiment_set_3_replicate_7 Factor:2 μl RAW_FILE_NAME=Liver NEG1.mzdata.xml SUBJECT_SAMPLE_FACTORS - ANP_experiment_set_3_replicate_8 Factor:2 μl RAW_FILE_NAME=Liver NEG2.mzdata.xml SUBJECT_SAMPLE_FACTORS - ANP_experiment_set_3_replicate_1 Factor:4 μl RAW_FILE_NAME=liver1 ANPPOS MS.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 (-20C or colder) 80% methanol to extract CO:COLLECTION_SUMMARY metabolites. First, 1 ml of 80% methanol was added to the liver and incubated CO:COLLECTION_SUMMARY for 10 min at -20oC. Glass beads were added to the liver and then the liver was CO:COLLECTION_SUMMARY lysed by bead-beating for 45 s using a Tissuelyser cell disrupter (Qiagen). The CO:COLLECTION_SUMMARY lysate was incubated for 10 min at -20oC and centrifuged (13200 rpm, 5 min) to CO:COLLECTION_SUMMARY separate metabolites from macromolecules. The supernatant was collected and 200 CO:COLLECTION_SUMMARY µl of 80% methanol was added to the pellet. The incubation, shaking and CO:COLLECTION_SUMMARY centrifugation steps were repeated twice to extract more metabolites from the CO:COLLECTION_SUMMARY pellet. The three supernatants were combined and centrifuged (14000 rpm, 10 min) CO:COLLECTION_SUMMARY to separate any remaining macromolecules from the metabolites. The combined CO:COLLECTION_SUMMARY supernatants were dried using a SpeedVac Concentrator (Savant, SPD131DDA) at CO:COLLECTION_SUMMARY 25oC and the dried metabolite samples were stored at -80oC. CO:SAMPLE_TYPE Liver CO:COLLECTION_METHOD 80% methanol CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY All samples were treated identically for this sub-study, except a higher volume TR:TREATMENT_SUMMARY (4 μl ANP_experiment_set_3_replicate_1) of one sample was injected than for the TR:TREATMENT_SUMMARY other samples (2 μl). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY On the day of metabolite analysis, dried-down extracts were reconstituted in 150 SP:SAMPLEPREP_SUMMARY µl 70% acetonitrile, at a relative protein concentration of ~ 2 µg/µl. Either SP:SAMPLEPREP_SUMMARY 4 µl (replicate 1) or 2 µl (all other replicates) of this reconstituted SP:SAMPLEPREP_SUMMARY extract was injected for LC/MS-based targeted and untargeted metabolite SP:SAMPLEPREP_SUMMARY profiling. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Tissue extracts were analyzed by LC/MS as described previously, using a platform CH:CHROMATOGRAPHY_SUMMARY comprised of an Agilent Model 1290 Infinity II liquid chromatography system CH:CHROMATOGRAPHY_SUMMARY coupled to an Agilent 6550 iFunnel time-of-flight MS analyzer. Chromatography of CH:CHROMATOGRAPHY_SUMMARY metabolites utilized aqueous normal phase (ANP) chromatography on a Diamond CH:CHROMATOGRAPHY_SUMMARY Hydride column (Microsolv). Mobile phases consisted of: (A) 50% isopropanol, CH:CHROMATOGRAPHY_SUMMARY containing 0.025% acetic acid, and (B) 90% acetonitrile containing 5 mM ammonium CH:CHROMATOGRAPHY_SUMMARY acetate. To eliminate the interference of metal ions on chromatographic peak CH:CHROMATOGRAPHY_SUMMARY integrity and electrospray ionization, EDTA was added to the mobile phase at a CH:CHROMATOGRAPHY_SUMMARY final concentration of 5 µM. The following gradient was applied: 0-1.0 min, 99% CH:CHROMATOGRAPHY_SUMMARY B; 1.0-15.0 min, to 20% B; 15.0 to 29.0, 0% B; 29.1 to 37min, 99% B. CH:INSTRUMENT_NAME Agilent Model 1290 Infinity II liquid chromatography system CH:COLUMN_NAME Cogent Diamond Hydride (150 × 2.1 mm, 4um) CH:COLUMN_TEMPERATURE 26 CH:FLOW_GRADIENT The following gradient was applied: 0-1.0 min, 99% B; 1.0-15.0 min, to 20% B; CH:FLOW_GRADIENT 15.0 to 29.0, 0% B; 29.1 to 37min, 99% B. CH:FLOW_RATE 0.3 mL/min CH:SOLVENT_A 50% isopropanol/50% water; 0.025% acetic acid CH:SOLVENT_B 90% acetonitrile/10% water; 5 mM ammonium acetate CH:CHROMATOGRAPHY_TYPE Normal phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE Other MS:MS_COMMENTS LC/MS-based targeted and untargeted metabolite profiling. For targeted analysis, MS:MS_COMMENTS raw LC/MS data was extracted by MassProfinder 8.0 (Agilent Technologies) using MS:MS_COMMENTS an in-house annotated personal metabolite database that contains 863 metabolites MS:MS_COMMENTS (Agilent Technologies). Additionally, molecular feature extraction (MFE) was MS:MS_COMMENTS performed for untargeted metabolite profiling using MassProfinder 8.0 (Agilent MS:MS_COMMENTS Technologies). The untargeted molecular features were imported into MassProfiler MS:MS_COMMENTS Professional 15.1 (MPP, Agilent Technologies) and searched against Metlin MS:MS_COMMENTS personal metabolite database (PCDL database 8.0), Human Metabolome Database MS:MS_COMMENTS (HMDB) and an in-house phospholipid database for tentative metabolite ID MS:MS_COMMENTS assignments, based on monoisotopic neutral mass (< 5 ppm mass accuracy) MS:MS_COMMENTS matches. Furthermore, a molecular formula generator (MFG) algorithm in MPP was MS:MS_COMMENTS used to generate and score empirical molecular formulae, based on a weighted MS:MS_COMMENTS consideration of monoisotopic mass accuracy, isotope abundance ratios, and MS:MS_COMMENTS spacing between isotope peaks. A tentative compound ID was assigned when PCDL MS:MS_COMMENTS database and MFG scores concurred for a given candidate molecule. Tentatively MS:MS_COMMENTS assigned molecules were reextracted using Profinder 8.0 for confirmation of MS:MS_COMMENTS untargeted results. Only non-lipid metabolites that were identified in study MS:MS_COMMENTS ST002349 were retained for this analysis. MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Ion abundance (peak area) MS_METABOLITE_DATA_START Samples ANP_experiment_set_3_replicate_2 ANP_experiment_set_3_replicate_3 ANP_experiment_set_3_replicate_4 ANP_experiment_set_3_replicate_5 ANP_experiment_set_3_replicate_6 ANP_experiment_set_3_replicate_7 ANP_experiment_set_3_replicate_8 ANP_experiment_set_3_replicate_1 Factors Factor:2 μl Factor:2 μl Factor:2 μl Factor:2 μl Factor:2 μl Factor:2 μl Factor:2 μl Factor:4 μl 1-Methylnicotinamide 806661.3453 247184.8542 13409.5490 3-Methylhistamine 382470.3498 4-Guanidinobutyric Acid 1773167.8580 2351788.3015 2346069.8860 2311130.4163 382016.4027 144765036.6480 5-Aminopentanoic Acid 185267852.7417 Acetylglutamic Acid Alanine 8168133.3332 94106.4333 92653.7602 81284.9731 5283625.0967 532056.9400 Arginine Argininosuccinic Acid 5151449.1896 Aspartic Acid Bilirubin 48667.0516 30541.3689 CAR 12:0 73322.4418 55192.8477 5968794.0372 CAR 14:1 365802.6054 219163.3367 226715.1465 183575.8700 187093.3340 222288.7909 CAR 16:0 2333375.6562 2127577.1374 2337239.8779 2307585.4836 965433.7273 6730200.6765 CAR 16:0;O 258865.2424 94434.9973 96237.4944 90991.2344 110672.7697 3641831.1517 CAR 16:1 845156.3561 702037.4155 798237.3594 802522.1443 489722.5817 2510342.2533 CAR 16:1;O 450648.1280 53296.9176 65860.1054 56694.1200 86009145.0912 CAR 18:0 1437114.8432 148340.5375 134812.7870 139112.3361 1140270.2729 793597.8474 CAR 18:1 4589134.7081 4578288.0740 4851458.0533 4877941.1404 2896936.8027 328460.3127 CAR 18:2 1927117.8899 1717697.0439 1855866.4399 1842997.1191 450258.2823 4975338.1848 CAR 18:3 227355.2292 53561.7956 49447.3192 83174.9655 3172670.1481 CAR 2:0 CAR 3:0 24906428.5419 46519994.8806 48491375.2202 47641282.0576 3228294.3868 7953022.5718 CAR 4:0 156179439.7749 160085182.3979 159698157.8508 3171456.6702 1504149.0890 CAR 4:1;O2 212049.9968 1390040.3821 1481802.8066 1493981.5715 126574.7083 508034.6755 CAR 5:0 10745315.3284 15070160.1077 15647113.1564 15434452.0338 1270926.7614 559430.4662 CAR 6:0 17475417.9831 28025506.8694 28640311.0682 28400197.3803 328282.5213 78294499.2211 Carnitine Choline 61923009.7056 60696635.0745 48743948.4543 Creatinine 4730044.5083 4140904.9778 4432119.6932 4413038.4028 293789.3138 Deoxycarnitine 8684185.5738 12062383.0770 12257132.4025 12042893.1049 2964972.1067 1509135.8847 Glutamine Guanidinosuccinic Acid 651117.6932 843297.8214 866778.8707 865409.6685 1865138.5807 2051421.8612 Guanine 1207312.2902 1821272.7117 1888981.0022 1914914.1548 952691.6119 12301793.9051 Hypoxanthine 48693269.4096 49927116.6072 374888292.5409 L-Monomethylarginine N1-Acetylspermidine 126396.2883 Nicotinamide 127923596.9529 131950201.1519 47849288.0934 Ornithine Phosphocholine 5473337.4487 7438022.2305 7645841.8223 7635409.2119 16964993.1385 58528579.5264 Proline Betaine 47593616.4877 48805936.3314 48822013.2522 226345.7089 1388230.4576 S-Adenosylmethionine Trimethyllysine 405705.1333 431647.6307 3724125.6935 UDP Urea 2593947.2299 4504459.5209 4654258.0232 2007386.5913 2135235.3964 Urocanic Acid MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant 1-Methylnicotinamide 457 20.3 3-Methylhistamine 69520 20.29 4-Guanidinobutyric Acid 500 9.14 5-Aminopentanoic Acid 12.91 Acetylglutamic Acid 8.84 Alanine 5950 7.31 Arginine 6322 22.01 Argininosuccinic Acid 16950 9.77 Aspartic Acid 5960 9.17 Bilirubin 5280352 1.07 CAR 12:0 7.95 CAR 14:1 7.68 CAR 16:0 7.56 CAR 16:0;O 7.95 CAR 16:1 7.58 CAR 16:1;O 7.97 CAR 18:0 7.4 CAR 18:1 7.39 CAR 18:2 7.46 CAR 18:3 7.5 CAR 2:0 11.58 CAR 3:0 10.8 CAR 4:0 10.07 CAR 4:1;O2 10.95 CAR 5:0 9.53 CAR 6:0 9.08 Carnitine 10917 12.05 Choline 305 20.29 Creatinine 588 5.09 Deoxycarnitine 134 14.96 Glutamine 5961 9.63 Guanidinosuccinic Acid 97856 7.74 Guanine 764 2.93 Hypoxanthine 790 2.48 L-Monomethylarginine 24.86 N1-Acetylspermidine 496 24.31 Nicotinamide 936 1.77 Ornithine 6262 16.8 Phosphocholine 1014 16.12 Proline Betaine 115244 9.11 S-Adenosylmethionine 23.72 Trimethyllysine 440121 24.26 UDP 6031 9.97 Urea 1176 2.6 Urocanic Acid 736715 6.516 METABOLITES_END #END