#METABOLOMICS WORKBENCH ftayyari1_20190514_084815 DATATRACK_ID:1724 STUDY_ID:ST001183 ANALYSIS_ID:AN001961 PROJECT_ID:PR000795 VERSION 1 CREATED_ON May 14, 2019, 6:57 pm #PROJECT PR:PROJECT_TITLE Correlations between LC-MS/MS-detected Glycomics and NMR- detected Metabolomics PR:PROJECT_TITLE in Caenorhabditis elegans Development. PR:PROJECT_SUMMARY New approach to evaluate the relationship between Caenorhabditis elegans PR:PROJECT_SUMMARY development, glycan abundance, and metabolites PR:INSTITUTE University of Georgia PR:DEPARTMENT Complex Carbohydrate Research Center PR:LABORATORY Edison PR:LAST_NAME Edison PR:FIRST_NAME Arthur PR:ADDRESS 315 Riverbend Road, Athens, GA, 30602, USA PR:EMAIL asedison@uga.edu PR:PHONE 706-542-8156 #STUDY ST:STUDY_TITLE Correlations between LC-MS/MS-detected Glycomics and NMR-detected Metabolomics ST:STUDY_TITLE in Caenorhabditis elegans Development. ST:STUDY_SUMMARY This study examines the relationship between glycans, metabolites, and ST:STUDY_SUMMARY development in C. elegans. Samples of N2 animals were synchronized and grown to ST:STUDY_SUMMARY five different time points that ranged from L1 to a mixed population of adults, ST:STUDY_SUMMARY gravid adults, and offspring. ST:INSTITUTE University of Georgia ST:DEPARTMENT Complex Carbohydrate Research Center ST:LABORATORY Edison and Wells ST:LAST_NAME Edison ST:FIRST_NAME Arthur ST:ADDRESS 315 Riverbend Road Athens, Georgia 30602-4712 USA ST:EMAIL aedison@uga.edu ST:PHONE 706-542-8156 #SUBJECT SU:SUBJECT_TYPE Other SU:SUBJECT_SPECIES Caenorhabditis elegans SU:TAXONOMY_ID 6239 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS L4/young adult W_09_FVP_N2_01_01 Sample_desc:Time point 3 SUBJECT_SAMPLE_FACTORS adults, gravid adults with mixed-stage offspring W_09_FVP_N2_07_02 Sample_desc:Time point 5 SUBJECT_SAMPLE_FACTORS adults, gravid adults with mixed-stage offspring W_09_FVP_N2_06_01 Sample_desc:Time point 5 SUBJECT_SAMPLE_FACTORS adults, gravid adults with mixed-stage offspring W_09_FVP_N2_08_04 Sample_desc:Time point 5 SUBJECT_SAMPLE_FACTORS adult W_09_FVP_N2_07_01 Sample_desc:Time point 4 SUBJECT_SAMPLE_FACTORS L2/L3 W_09_FVP_N2_08_03 Sample_desc:Time point 2 SUBJECT_SAMPLE_FACTORS Adults W_09_FVP_N2_06_04 Sample_desc:Time point 4 SUBJECT_SAMPLE_FACTORS L2/L3 W_09_FVP_N2_07_03 Sample_desc:Time point 2 SUBJECT_SAMPLE_FACTORS L4/young adult W_09_FVP_N2_05_01 Sample_desc:Time point 3 SUBJECT_SAMPLE_FACTORS adult W_09_FVP_N2_05_02 Sample_desc:Time point 4 SUBJECT_SAMPLE_FACTORS adult W_09_FVP_N2_08_02 Sample_desc:Time point 4 SUBJECT_SAMPLE_FACTORS L4/young adult W_09_FVP_N2_04_01 Sample_desc:Time point 3 SUBJECT_SAMPLE_FACTORS L4/young adult W_09_FVP_N2_07_04 Sample_desc:Time point 3 SUBJECT_SAMPLE_FACTORS L4/young adult W_09_FVP_N2_06_03 Sample_desc:Time point 3 SUBJECT_SAMPLE_FACTORS adults, gravid adults with mixed-stage offspring W_09_FVP_N2_04_02 Sample_desc:Time point 5 SUBJECT_SAMPLE_FACTORS adults, gravid adults with mixed-stage offspring W_09_FVP_N2_01_04 Sample_desc:Time point 5 SUBJECT_SAMPLE_FACTORS L2/L3 W_09_FVP_N2_03_01 Sample_desc:Time point 2 SUBJECT_SAMPLE_FACTORS L2/L3 W_09_FVP_N2_05_04 Sample_desc:Time point 2 SUBJECT_SAMPLE_FACTORS adult W_09_FVP_N2_04_04 Sample_desc:Time point 4 SUBJECT_SAMPLE_FACTORS L4/young adult W_09_FVP_N2_08_01 Sample_desc:Time point 3 SUBJECT_SAMPLE_FACTORS adult W_09_FVP_N2_03_03 Sample_desc:Time point 4 SUBJECT_SAMPLE_FACTORS L2/L3 W_09_FVP_N2_06_02 Sample_desc:Time point 2 SUBJECT_SAMPLE_FACTORS adults, gravid adults with mixed-stage offspring W_09_FVP_N2_03_04 Sample_desc:Time point 5 SUBJECT_SAMPLE_FACTORS L2/L3 W_09_FVP_N2_01_02 Sample_desc:Time point 2 SUBJECT_SAMPLE_FACTORS L4/young adult W_09_FVP_N2_03_02 Sample_desc:Time point 3 SUBJECT_SAMPLE_FACTORS L2/L3 W_09_FVP_N2_04_03 Sample_desc:Time point 2 SUBJECT_SAMPLE_FACTORS adult W_09_FVP_N2_01_03 Sample_desc:Time point 4 SUBJECT_SAMPLE_FACTORS adults, gravid adults with mixed-stage offspring W_09_FVP_N2_05_03 Sample_desc:Time point 5 SUBJECT_SAMPLE_FACTORS L1 W_09_FVP_N2_L1s Sample_desc:Time point 1 #COLLECTION CO:COLLECTION_SUMMARY This study used N2, the laboratory reference strain of C. elegans, which was CO:COLLECTION_SUMMARY obtained from the Caenorhabditis Genetics Center (CGC). We followed the general CO:COLLECTION_SUMMARY protocol published previously for obtaining liquid cultures of synchronized CO:COLLECTION_SUMMARY worms. This defines our biological replicate: A single L1 animal from a CO:COLLECTION_SUMMARY synchronized culture was placed onto an agar plate seeded with E. coli MG1655. CO:COLLECTION_SUMMARY This plate was grown until there were a large number of young gravid adult CO:COLLECTION_SUMMARY hermaphrodites (about 48 h at 24 °C. The plate was then washed into a 15 mL CO:COLLECTION_SUMMARY tube with M9 buffer, rinsed 3x with M9, and lysed with an alkaline hypochlorite CO:COLLECTION_SUMMARY solution until about 50% of the worms were dissolved (no more than 5 min). Then, CO:COLLECTION_SUMMARY M9 buffer was added to dilute the lysing solution, and the liquid was removed CO:COLLECTION_SUMMARY after gentle centrifugation at 580 g for 2 min to pellet the eggs without CO:COLLECTION_SUMMARY breaking them. This step was repeated 3x to completely remove the lysis CO:COLLECTION_SUMMARY solution. After the final rinse, eggs were resuspended in sterile water before a CO:COLLECTION_SUMMARY sucrose gradient to remove cellular debris and bacteria. An equal volume (5 mL) CO:COLLECTION_SUMMARY of 60% sucrose was added to the eggs in water and centrifuged at 350 g for 4 CO:COLLECTION_SUMMARY min. The eggs were rinsed to remove residual sucrose and once they hatched, CO:COLLECTION_SUMMARY approximately 200,000 animals were transferred to 20 mL of S-complete with 2 mL CO:COLLECTION_SUMMARY of 50% MG1655. This material was grown to the desired developmental stage and CO:COLLECTION_SUMMARY prepared as described below. The C. elegans cultures were synchronized, but they CO:COLLECTION_SUMMARY gradually lost synchrony over time. We collected samples at 5 different time CO:COLLECTION_SUMMARY points (T1-T5) in development. We report results using these time points rather CO:COLLECTION_SUMMARY than developmental larval stages, since they are not all pure stage cultures. CO:COLLECTION_SUMMARY The first time, T1, was collected immediately after hatching and was perfectly CO:COLLECTION_SUMMARY synchronized L1 animals, but as time progressed the cultures became more mixed. CO:COLLECTION_SUMMARY The other samples were collected at 22, 36, 49, and 90 hours (T2, T3, T4, and CO:COLLECTION_SUMMARY T5, respectively) after feeding the cultures. T5 was a mixture of adults, gravid CO:COLLECTION_SUMMARY adults, and offspring. Each of the five time points were replicated seven times. CO:COLLECTION_SUMMARY Stage-specific information can be recovered, even with samples that have lost CO:COLLECTION_SUMMARY synchrony. CO:SAMPLE_TYPE Worms #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The remaining 97.5% of the worm pellets after biosorting were bead homogenized SP:SAMPLEPREP_SUMMARY with 80% methanol/20% water and remaining pellets after extraction used for SP:SAMPLEPREP_SUMMARY glycomics. SP:SAMPLEPREP_PROTOCOL_FILENAME NMR_sample_preparation, Glycomics_sample preparation #ANALYSIS AN:OPERATOR_NAME Fariba Tayyari #NMR NM:INSTRUMENT_NAME Bruker AVIII-HD NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:NMR_COMMENTS 1D NMR for all the samples and 2D NMR on selected samples NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_SOLVENT D2O NM:NMR_TUBE_SIZE 5mm x 7 in NM:SHIMMING_METHOD topshim NM:TEMPERATURE 27 NM:NUMBER_OF_SCANS 128 NM:DUMMY_SCANS 4 NM:ACQUISITION_TIME 2.7198913 NM:SPECTRAL_WIDTH 20.0276 NM:LINE_BROADENING 0.3 #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS N/A NMR_METABOLITE_DATA_START N/A NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name METABOLITES_END #END