#METABOLOMICS WORKBENCH nemes_20180808_130309 DATATRACK_ID:1469 STUDY_ID:ST001032 ANALYSIS_ID:AN001692 PROJECT_ID:PR000690 VERSION 1 CREATED_ON August 9, 2018, 3:10 pm #PROJECT PR:PROJECT_TITLE Single-cell Profiling of Cationic and Anionic Metabolites in Live Frog (Xenopus) PR:PROJECT_TITLE Embryos using Microprobe Capillary Electrophoresis Mass Spectrometry PR:PROJECT_TYPE Metabolic profiling of anionic and cationic metabolites in single cells PR:PROJECT_SUMMARY The goal of this study was to enable the analysis of anionic and cationic PR:PROJECT_SUMMARY metabolites from the same identified single cell in Xenopus laevis embryos. A 10 PR:PROJECT_SUMMARY nL portion of identified animal-ventral (V1) cells was aspirated from 8-cell PR:PROJECT_SUMMARY embryos, and metabolites were extracted from the aspirate, before PR:PROJECT_SUMMARY characterization of cationic and anionic compounds using a custom-built PR:PROJECT_SUMMARY capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry PR:PROJECT_SUMMARY platform. A total of ~250 cationic molecular features and ~150 anionic molecular PR:PROJECT_SUMMARY features were detected, including 76 metabolites that were identified in this PR:PROJECT_SUMMARY study. Pathway analysis of the identified metabolites highlighted PR:PROJECT_SUMMARY arginine-proline metabolism of significance. PR:INSTITUTE University of Maryland PR:DEPARTMENT Department of Chemistry & Biochemistry PR:LABORATORY Nemes Laboratory PR:LAST_NAME Nemes PR:FIRST_NAME Peter PR:ADDRESS 0107 Chemistry Building, 8051 Regents Dr, College Park, MD 20742 PR:EMAIL nemes@umd.edu PR:PHONE 301-405-0373 PR:FUNDING_SOURCE National Cancer Institute grant 7R03CA211635 #STUDY ST:STUDY_TITLE Single-cell Profiling of Cationic and Anionic Metabolites in Live Frog (Xenopus) ST:STUDY_TITLE Embryos using Microprobe Capillary Electrophoresis Mass Spectrometry ST:STUDY_TYPE Metabolic profiling of single cells ST:STUDY_SUMMARY The goal of this study was to enable the analysis of anionic and cationic ST:STUDY_SUMMARY metabolites from the same identified single cell in Xenopus laevis embryos. A 10 ST:STUDY_SUMMARY nL portion of identified animal-ventral (V1) cells was aspirated from 8-cell ST:STUDY_SUMMARY embryos, and metabolites were extracted from the aspirate, before ST:STUDY_SUMMARY characterization of cationic and anionic compounds using a custom-built ST:STUDY_SUMMARY capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry ST:STUDY_SUMMARY platform. A total of ~250 cationic molecular features and ~150 anionic molecular ST:STUDY_SUMMARY features were detected, including 76 metabolites that were identified in this ST:STUDY_SUMMARY study. Pathway analysis of the identified metabolites highlighted ST:STUDY_SUMMARY arginine-proline metabolism of significance. ST:INSTITUTE University of Maryland ST:DEPARTMENT Department of Chemistry & Biochemistry ST:LABORATORY Nemes Laboratory ST:LAST_NAME Nemes ST:FIRST_NAME Peter ST:ADDRESS 0107 Chemistry Building 8051 Regents Drive ST:EMAIL nemes@umd.edu ST:PHONE 3014050373 ST:NUM_GROUPS 4 biological replicates (each different cell from a different embryo) + 1-to-2 ST:NUM_GROUPS technical replicates (same extract analyzed multiple times) ST:TOTAL_SUBJECTS 4 different V1 cells were analyzed, each from a different embryo #SUBJECT SU:SUBJECT_TYPE Other SU:SUBJECT_SPECIES Xenopus laevis SU:TAXONOMY_ID 8355 SU:AGE_OR_AGE_RANGE Embryos were obtained from natural mating of frogs (Nasco) SU:WEIGHT_OR_WEIGHT_RANGE Sexually mature male and female frogs SU:GENDER Not applicable #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 2016-12-20_EP02 negative mode 8-cell embryo1 V1E1T1N Embryo Type:WT Biological Replicate Number=1 SUBJECT_SAMPLE_FACTORS 2017-07-31_EP02 negative mode 8-cell embryo2.d V1E2T1N Embryo Type:WT Biological Replicate Number=2 SUBJECT_SAMPLE_FACTORS 2017-08-01_EP02 positive mode 8-cell embryo2.d V1E2T1P Embryo Type:WT Biological Replicate Number=2 SUBJECT_SAMPLE_FACTORS 2017-11-17_EP04 positive mode V1-E3.d V1E3T1P Embryo Type:WT Biological Replicate Number=3 SUBJECT_SAMPLE_FACTORS 2017-11-27_EP02 negative mode V1-E4.d V1E4T1N Embryo Type:WT Biological Replicate Number=4 SUBJECT_SAMPLE_FACTORS 2017-11-17_EP03 positive mode V1-E3-TR2.d V1E3T2P Embryo Type:WT Biological Replicate Number=3 #COLLECTION CO:COLLECTION_SUMMARY Cells were identified based on morphology, pigmentation, and location in the CO:COLLECTION_SUMMARY embryo in comparison to established cell-fate maps for Xenopus laevis embryos. A CO:COLLECTION_SUMMARY portion of the identified V1 cell was microaspirated using a fabricated CO:COLLECTION_SUMMARY microcapillary. CO:COLLECTION_PROTOCOL_ID Portero 2018 Metabolomics Workbench Protocols FINAL 2018-08-08 CO:COLLECTION_PROTOCOL_FILENAME nemes_20180725_143242_PR_SP_Liu_2018_Metabolomics_Workbench_Protocol.pdf CO:SAMPLE_TYPE embryonic cell CO:COLLECTION_METHOD Microaspiration of cell content CO:COLLECTION_FREQUENCY 1 collection per cell CO:COLLECTION_DURATION 5 s for aspiration CO:VOLUMEORAMOUNT_COLLECTED Ca. 10 nL per aspiration CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY All protocols related to the handling and manipulation of animals were approved TR:TREATMENT_SUMMARY by the University of Maryland, College Park (College Park, MD). Embryos were TR:TREATMENT_SUMMARY dejellied using 2% cystine solution, cultured in 100% Steinberg's solution TR:TREATMENT_SUMMARY (media), and used without further treatment. TR:TREATMENT_PROTOCOL_ID IACUC # R-DEC-17-57 TR:TREATMENT_PROTOCOL_FILENAME nemes_20180725_143242_PR_SP_Liu_2018_Metabolomics_Workbench_Protocol.pdf #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Ca. 10 nL of cell content were aspirated from identified cells. The aspirated SP:SAMPLEPREP_SUMMARY material was ejected into ~4 uL of aqueous mixture of 40% acetonitrile and 40% SP:SAMPLEPREP_SUMMARY methanol to extract metabolites. SP:SAMPLEPREP_PROTOCOL_FILENAME nemes_20180725_143242_PR_SP_Liu_2018_Metabolomics_Workbench_Protocol.pdf SP:PROCESSING_METHOD On ice, then extracts stored at -80 degC until analysis. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACTION_METHOD In cold aqueous mixture of 40% acetonitrile and 40% methanol. SP:EXTRACT_ENRICHMENT none SP:EXTRACT_CLEANUP none SP:EXTRACT_STORAGE -80℃ SP:SUBCELLULAR_LOCATION Unknown #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Metabolites were separated in a custom-built capillary electrophoresis (CE) CH:CHROMATOGRAPHY_SUMMARY system. CH:CHROMATOGRAPHY_TYPE CE CH:INSTRUMENT_NAME Custom-built CE system CH:COLUMN_NAME Bare fused silica capillary CH:COLUMN_TEMPERATURE Room temperature CH:SOLVENT_A During cationic separation, the background electrolyte used in postive CH:SOLVENT_A ionization mode was 1% formic acid in water (isocratic). CH:INJECTION_TEMPERATURE Room temperature CH:SAMPLE_INJECTION Ca. 10 nL CH:ANALYTICAL_TIME 45 min of separation CH:CAPILLARY_VOLTAGE During cationic separation, +19,000-20,000 V was applied on the inlet end of the CH:CAPILLARY_VOLTAGE CE capillary. CH:PRECONDITIONING Sodium hydroxide solution CH:SHEATH_LIQUID During cationic analysis, the electrospray sheath solution was 50% methanol with CH:SHEATH_LIQUID 0.1% formic acid. #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Nemes Laboratory AN:OPERATOR_NAME Erika Portero AN:SOFTWARE_VERSION Compass 4.3 #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Bruker Impact HD MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:CAPILLARY_TEMPERATURE 100 degC MS:CAPILLARY_VOLTAGE +1700 V during cationic analysis MS:MASS_ACCURACY <5 ppm MS:DATAFORMAT mzML MS:SCANNING_CYCLE 2 Hz MS:SCANNING_RANGE mz 50-550 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples V1E2T1P V1E3T1P V1E3T2P Factors Embryo Type:WT Embryo Type:WT Embryo Type:WT Lysine 18719536 7011901 Arginine 16969458 34194288 Guanine 19523818 179979696 Pyridoxal 2847430 678385 1816902 Creatine 1315571 Alanine Asparagine Glutamine 25867 55428524 Glutamate 278801 256206176 Aspartic acid 31290716 66008124 Hypoxanthine 154605728 1385160 Hydroxyproline 11474813 98667 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Moverz Quant Local ID (CAS) PubChem CID Lysine 16.4 5962 Arginine 17.08 6322 Guanine 32.1 764 Pyridoxal 34.1 1050 Creatine 20.94 586 Alanine 32.1 5950 Asparagine 26.75 6267 Glutamine 27.03 5961 Glutamate 27.09 33032 Aspartic acid 29.99 5960 Hypoxanthine 65.4 7090 Hydroxyproline 32.57 5810 METABOLITES_END #END