#METABOLOMICS WORKBENCH wernerajz_20190302_125834_mwtab.txt DATATRACK_ID:1662 STUDY_ID:ST001199 ANALYSIS_ID:AN001995 PROJECT_ID:PR000808 VERSION 1 CREATED_ON June 18, 2019, 9:51 pm #PROJECT PR:PROJECT_TITLE A comprehensive time-course metabolite profiling of the model cyanobacterium PR:PROJECT_TITLE Synechocystis sp. PCC 6803 under diurnal light:dark cycles PR:PROJECT_SUMMARY Cyanobacteria are a model photoautotroph and a chassis for the sustainable PR:PROJECT_SUMMARY production of fuels and chemicals. Yet, knowledge of photoautotrophic metabolism PR:PROJECT_SUMMARY in the natural environment of day/night cycles is lacking yet has implications PR:PROJECT_SUMMARY for improved yield from plants, algae, and cyanobacteria. Here, a thorough PR:PROJECT_SUMMARY approach to characterizing diverse metabolites—including carbohydrates, PR:PROJECT_SUMMARY lipids, amino acids, pigments, co-factors, nucleic acids and PR:PROJECT_SUMMARY polysaccharides—in the model cyanobacterium Synechocystis sp. PCC 6803 (S. PR:PROJECT_SUMMARY 6803) under sinusoidal diurnal light-dark cycles was developed and applied. A PR:PROJECT_SUMMARY custom photobioreactor and novel multi-platform mass spectrometry workflow PR:PROJECT_SUMMARY enabled metabolite profiling every 30-120 minutes across a 24-hour diurnal PR:PROJECT_SUMMARY sinusoidal LD (“sinLD”) cycle peaking at 1,600 mol photons m 2 s-1. We PR:PROJECT_SUMMARY report widespread oscillations across the sinLD cycle with 90%, 94%, and 40% of PR:PROJECT_SUMMARY the identified polar/semi-polar, non-polar, and polymeric metabolites displaying PR:PROJECT_SUMMARY statistically significant oscillations, respectively. Microbial growth displayed PR:PROJECT_SUMMARY distinct lag, biomass accumulation, and cell division phases of growth. During PR:PROJECT_SUMMARY the lag phase, amino acids (AA) and nucleic acids (NA) accumulated to high PR:PROJECT_SUMMARY levels per cell followed by decreased levels during the biomass accumulation PR:PROJECT_SUMMARY phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates PR:PROJECT_SUMMARY displayed sharp oscillations per cell at the day-to-night transition. Potential PR:PROJECT_SUMMARY bottlenecks in central carbon metabolism are highlighted. Together, this report PR:PROJECT_SUMMARY provides a comprehensive view of photosynthetic metabolite behavior with high PR:PROJECT_SUMMARY temporal resolution, offering insight into the impact of growth synchronization PR:PROJECT_SUMMARY to light cycles via circadian rhythms. Incorporation into computational modeling PR:PROJECT_SUMMARY and metabolic engineering efforts promises to improve industrially-relevant PR:PROJECT_SUMMARY strain design. PR:INSTITUTE Colorado State University PR:DEPARTMENT Chemical and PR:LAST_NAME Peebles PR:FIRST_NAME Christie PR:ADDRESS 700 Meridian Ave, Fort Collins, CO 80523 USA PR:EMAIL wernerajz@gmail.com PR:PHONE 2699981811 #STUDY ST:STUDY_TITLE Non-targeted LC-MS Analysis of Soluble Metabolites in the Non-Polar MTBE Phase ST:STUDY_TITLE (part-V) ST:STUDY_SUMMARY Cyanobacteria are a model photoautotroph and a chassis for the sustainable ST:STUDY_SUMMARY production of fuels and chemicals. Yet, knowledge of photoautotrophic metabolism ST:STUDY_SUMMARY in the natural environment of day/night cycles is lacking yet has implications ST:STUDY_SUMMARY for improved yield from plants, algae, and cyanobacteria. Here, a thorough ST:STUDY_SUMMARY approach to characterizing diverse metabolites—including carbohydrates, ST:STUDY_SUMMARY lipids, amino acids, pigments, co-factors, nucleic acids and ST:STUDY_SUMMARY polysaccharides—in the model cyanobacterium Synechocystis sp. PCC 6803 (S. ST:STUDY_SUMMARY 6803) under sinusoidal diurnal light-dark cycles was developed and applied. A ST:STUDY_SUMMARY custom photobioreactor and novel multi-platform mass spectrometry workflow ST:STUDY_SUMMARY enabled metabolite profiling every 30-120 minutes across a 24-hour diurnal ST:STUDY_SUMMARY sinusoidal LD (“sinLD”) cycle peaking at 1,600 mol photons m 2 s-1. We ST:STUDY_SUMMARY report widespread oscillations across the sinLD cycle with 90%, 94%, and 40% of ST:STUDY_SUMMARY the identified polar/semi-polar, non-polar, and polymeric metabolites displaying ST:STUDY_SUMMARY statistically significant oscillations, respectively. Microbial growth displayed ST:STUDY_SUMMARY distinct lag, biomass accumulation, and cell division phases of growth. During ST:STUDY_SUMMARY the lag phase, amino acids (AA) and nucleic acids (NA) accumulated to high ST:STUDY_SUMMARY levels per cell followed by decreased levels during the biomass accumulation ST:STUDY_SUMMARY phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates ST:STUDY_SUMMARY displayed sharp oscillations per cell at the day-to-night transition. Potential ST:STUDY_SUMMARY bottlenecks in central carbon metabolism are highlighted. Together, this report ST:STUDY_SUMMARY provides a comprehensive view of photosynthetic metabolite behavior with high ST:STUDY_SUMMARY temporal resolution, offering insight into the impact of growth synchronization ST:STUDY_SUMMARY to light cycles via circadian rhythms. Incorporation into computational modeling ST:STUDY_SUMMARY and metabolic engineering efforts promises to improve industrially-relevant ST:STUDY_SUMMARY strain design. ST:INSTITUTE Colorado State University ST:DEPARTMENT Chemical and Biological Engineering ST:LAST_NAME Peebles ST:FIRST_NAME Christie ST:ADDRESS 700 Meridian Ave, Fort Collins, CO 80523 ST:EMAIL christie.peebles@colostate.edu ST:PHONE 970-491-6779 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Synechocystis sp. PCC 6803 SU:TAXONOMY_ID 1148 SU:GENOTYPE_STRAIN NCBI:txid1148 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1-Synechocystis_6803-cell-0-1 time:-2 SUBJECT_SAMPLE_FACTORS - 2-Synechocystis_6803-cell-0-2 time:-2 SUBJECT_SAMPLE_FACTORS - 3-Synechocystis_6803-cell-0-3 time:-2 SUBJECT_SAMPLE_FACTORS - 4-Synechocystis_6803-cell-1-1 time:-1 SUBJECT_SAMPLE_FACTORS - 5-Synechocystis_6803-cell-1-2 time:-1 SUBJECT_SAMPLE_FACTORS - 6-Synechocystis_6803-cell-1-3 time:-1 SUBJECT_SAMPLE_FACTORS - 7-Synechocystis_6803-cell-1.5-1 time:-0.5 SUBJECT_SAMPLE_FACTORS - 8-Synechocystis_6803-cell-1.5-2 time:-0.5 SUBJECT_SAMPLE_FACTORS - 9-Synechocystis_6803-cell-1.5-3 time:-0.5 SUBJECT_SAMPLE_FACTORS - 10-Synechocystis_6803-cell-2-1 time:- SUBJECT_SAMPLE_FACTORS - 11-Synechocystis_6803-cell-2-2 time:- SUBJECT_SAMPLE_FACTORS - 12-Synechocystis_6803-cell-2-3 time:- SUBJECT_SAMPLE_FACTORS - 13-Synechocystis_6803-cell-2.5-1 time:0.5 SUBJECT_SAMPLE_FACTORS - 14-Synechocystis_6803-cell-2.5-2 time:0.5 SUBJECT_SAMPLE_FACTORS - 15-Synechocystis_6803-cell-2.5-3 time:0.5 SUBJECT_SAMPLE_FACTORS - 16-Synechocystis_6803-cell-3-1 time:1 SUBJECT_SAMPLE_FACTORS - 17-Synechocystis_6803-cell-3-2 time:1 SUBJECT_SAMPLE_FACTORS - 18-Synechocystis_6803-cell-3-3 time:1 SUBJECT_SAMPLE_FACTORS - 19-Synechocystis_6803-cell-4-1 time:2 SUBJECT_SAMPLE_FACTORS - 20-Synechocystis_6803-cell-4-2 time:2 SUBJECT_SAMPLE_FACTORS - 21-Synechocystis_6803-cell-4-3 time:2 SUBJECT_SAMPLE_FACTORS - 22-Synechocystis_6803-cell-6-1 time:4 SUBJECT_SAMPLE_FACTORS - 23-Synechocystis_6803-cell-6-2 time:4 SUBJECT_SAMPLE_FACTORS - 24-Synechocystis_6803-cell-6-3 time:4 SUBJECT_SAMPLE_FACTORS - 25-Synechocystis_6803-cell-8-1 time:6 SUBJECT_SAMPLE_FACTORS - 26-Synechocystis_6803-cell-8-2 time:6 SUBJECT_SAMPLE_FACTORS - 27-Synechocystis_6803-cell-8-3 time:6 SUBJECT_SAMPLE_FACTORS - 28-Synechocystis_6803-cell-10-1 time:8 SUBJECT_SAMPLE_FACTORS - 29-Synechocystis_6803-cell-10-2 time:8 SUBJECT_SAMPLE_FACTORS - 30-Synechocystis_6803-cell-10-3 time:8 SUBJECT_SAMPLE_FACTORS - 31-Synechocystis_6803-cell-12-1 time:10 SUBJECT_SAMPLE_FACTORS - 32-Synechocystis_6803-cell-12-2 time:10 SUBJECT_SAMPLE_FACTORS - 33-Synechocystis_6803-cell-12-3 time:10 SUBJECT_SAMPLE_FACTORS - 34-Synechocystis_6803-cell-13-1 time:11 SUBJECT_SAMPLE_FACTORS - 35-Synechocystis_6803-cell-13-2 time:11 SUBJECT_SAMPLE_FACTORS - 36-Synechocystis_6803-cell-13-3 time:11 SUBJECT_SAMPLE_FACTORS - 37-Synechocystis_6803-cell-13.5-1 time:11.5 SUBJECT_SAMPLE_FACTORS - 38-Synechocystis_6803-cell-13.5-2 time:11.5 SUBJECT_SAMPLE_FACTORS - 39-Synechocystis_6803-cell-13.5-3 time:11.5 SUBJECT_SAMPLE_FACTORS - 40-Synechocystis_6803-cell-14-1 time:12 SUBJECT_SAMPLE_FACTORS - 41-Synechocystis_6803-cell-14-2 time:12 SUBJECT_SAMPLE_FACTORS - 42-Synechocystis_6803-cell-14-3 time:12 SUBJECT_SAMPLE_FACTORS - 43-Synechocystis_6803-cell-14.5-1 time:12.5 SUBJECT_SAMPLE_FACTORS - 44-Synechocystis_6803-cell-14.5-2 time:12.5 SUBJECT_SAMPLE_FACTORS - 45-Synechocystis_6803-cell-14.5-3 time:12.5 SUBJECT_SAMPLE_FACTORS - 46-Synechocystis_6803-cell-15-1 time:13 SUBJECT_SAMPLE_FACTORS - 47-Synechocystis_6803-cell-15-2 time:13 SUBJECT_SAMPLE_FACTORS - 48-Synechocystis_6803-cell-15-3 time:13 SUBJECT_SAMPLE_FACTORS - 49-Synechocystis_6803-cell-16-1 time:14 SUBJECT_SAMPLE_FACTORS - 50-Synechocystis_6803-cell-16-2 time:14 SUBJECT_SAMPLE_FACTORS - 51-Synechocystis_6803-cell-16-3 time:14 SUBJECT_SAMPLE_FACTORS - 52-Synechocystis_6803-cell-18-1 time:16 SUBJECT_SAMPLE_FACTORS - 53-Synechocystis_6803-cell-18-2 time:16 SUBJECT_SAMPLE_FACTORS - 54-Synechocystis_6803-cell-18-3 time:16 SUBJECT_SAMPLE_FACTORS - 55-Synechocystis_6803-cell-20-1 time:18 SUBJECT_SAMPLE_FACTORS - 56-Synechocystis_6803-cell-20-2 time:18 SUBJECT_SAMPLE_FACTORS - 57-Synechocystis_6803-cell-20-3 time:18 SUBJECT_SAMPLE_FACTORS - 58-Synechocystis_6803-cell-22-1 time:20 SUBJECT_SAMPLE_FACTORS - 59-Synechocystis_6803-cell-22-2 time:20 SUBJECT_SAMPLE_FACTORS - 60-Synechocystis_6803-cell-22-3 time:20 SUBJECT_SAMPLE_FACTORS - 61-Synechocystis_6803-cell-24-1 time:22 SUBJECT_SAMPLE_FACTORS - 62-Synechocystis_6803-cell-24-2 time:22 SUBJECT_SAMPLE_FACTORS - 63-Synechocystis_6803-cell-24-3 time:22 SUBJECT_SAMPLE_FACTORS - 64-Synechocystis_6803-cell-25-1 time:23 SUBJECT_SAMPLE_FACTORS - 65-Synechocystis_6803-cell-25-2 time:23 SUBJECT_SAMPLE_FACTORS - 66-Synechocystis_6803-cell-25-3 time:23 SUBJECT_SAMPLE_FACTORS - 67-Synechocystis_6803-cell-25.5-1 time:23.5 SUBJECT_SAMPLE_FACTORS - 70-Synechocystis_6803-cell-26-1 time:24 SUBJECT_SAMPLE_FACTORS - 71-Synechocystis_6803-cell-26-2 time:24 SUBJECT_SAMPLE_FACTORS - 72-Synechocystis_6803-cell-26-3 time:24 SUBJECT_SAMPLE_FACTORS - QC1 time:26 SUBJECT_SAMPLE_FACTORS - QC2 time:26 SUBJECT_SAMPLE_FACTORS - QC3 time:26 SUBJECT_SAMPLE_FACTORS - QC4 time:26 SUBJECT_SAMPLE_FACTORS - QC5 time:26 SUBJECT_SAMPLE_FACTORS - QC6 time:26 SUBJECT_SAMPLE_FACTORS - QC7 time:26 SUBJECT_SAMPLE_FACTORS - QC8 time:26 SUBJECT_SAMPLE_FACTORS - QC9 time:26 SUBJECT_SAMPLE_FACTORS - QC10 time:26 SUBJECT_SAMPLE_FACTORS - QC11 time:26 SUBJECT_SAMPLE_FACTORS - QC12 time:26 SUBJECT_SAMPLE_FACTORS - QC13 time:26 #COLLECTION CO:COLLECTION_SUMMARY For each metabolomics time-point, a 10 mL culture were rapidly sampled via CO:COLLECTION_SUMMARY sterile on-reactor syringes into a pre-weighed centrifuge tube, quenched in CO:COLLECTION_SUMMARY -4°C 1X PBS, spun at 3,000g for 5 min., decanted, frozen in liquid nitrogen, CO:COLLECTION_SUMMARY and lyophilized at -50°C. The workflow from sampling to centrifugation took < 2 CO:COLLECTION_SUMMARY minutes; lyophilized samples were stored at -80°C for < 1 month prior to CO:COLLECTION_SUMMARY extraction. A biphasic extraction from lyophilized cell pellets was performed CO:COLLECTION_SUMMARY via a 2:1:1.6 MTBE:MeOH:H2O biphasic extraction, modified from the protocol CO:COLLECTION_SUMMARY developed by Salem et al. (Salem et al., 2016) resulting in a top layer of MTBE CO:COLLECTION_SUMMARY with non-polar soluble metabolites, a lower layer of MeOH:H2O with polar and CO:COLLECTION_SUMMARY semi-polar soluble metabolites, and an insoluble pellet. Each liquid layer was CO:COLLECTION_SUMMARY transferred to a fresh glass vial and dried under nitrogen gas overnight. The CO:COLLECTION_SUMMARY MTBE layer was resuspended in 1:1 toluene:MeOH and analyzed via Q-TOF-MS with a CO:COLLECTION_SUMMARY UPLC Phenyl Hexyl column (“RP-MS”). The MeOH:H2O layer was resuspended in CO:COLLECTION_SUMMARY 1:1 H2O:MeOH, split evenly and subjected to either i) derivatization in CO:COLLECTION_SUMMARY methoxyamine HCl and MSTFA followed by GC-MS analysis, or ii) targeted SRM CO:COLLECTION_SUMMARY analysis on a tandem quadrupole-MS equipped with a HILIC column. The insoluble CO:COLLECTION_SUMMARY pellet was hydrolyzed with a hydrochloric acid (HCl) based on previously CO:COLLECTION_SUMMARY published protocols (Fountoulakis and Lahm, 1998) (Huang, Kaiser and Benner, CO:COLLECTION_SUMMARY 2012) to analyze individual amino acids, nucleoside, and carbohydrate content of CO:COLLECTION_SUMMARY the insoluble polymers utilizing MTBSTFA derivatization for insoluble amino CO:COLLECTION_SUMMARY acids. Of the soluble phases, 10 µL were removed from each sample and pooled to CO:COLLECTION_SUMMARY create a QC sample, mixed, and aliquoted into thirteen vials. A QC sample was CO:COLLECTION_SUMMARY run after every sixth injection. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY Synechocystis sp. PCC 6803 [N-1] (ATCC 27184, NCBI Taxonomy ID: 1080229) was TR:TREATMENT_SUMMARY utilized for all experiments. A light-emitting diode photobioreactor (LED PBR) TR:TREATMENT_SUMMARY was engineered to provide a rectified sinusoidal waveform light profile which TR:TREATMENT_SUMMARY (results in the negative half-cycle being set to zero) via two custom 4000K TR:TREATMENT_SUMMARY White LED panels (Reliance Laboratories, Port Townsend WA) arranged opposite a TR:TREATMENT_SUMMARY water bath facing inwards, 5% CO2 at 200 mL min-1 via in-house gas mixing and TR:TREATMENT_SUMMARY custom aerators to provide sufficient mixing, 27-30°C temperature control via a TR:TREATMENT_SUMMARY Huber Ministat and custom water bath (Midwest Custom Aquarium, Starbuck MN), and TR:TREATMENT_SUMMARY improved light penetration at high volumes via custom flat-panel reactors (FPRs) TR:TREATMENT_SUMMARY built in a circular geometry to maximize mixing (Allen Scientific Glass, Boulder TR:TREATMENT_SUMMARY CO) (Figure S1). At the peak, 1,600 mol photons m-2s-1 (E) was provided as TR:TREATMENT_SUMMARY measured by LightScout Quantum Meter (Model: 3415FXSE). . A single LED-PBR was TR:TREATMENT_SUMMARY inoculated and entrained to sinLD cycles for two days; this entrained culture TR:TREATMENT_SUMMARY was then use inoculated three biological triplicate FPRs in the LED PBR (Figure TR:TREATMENT_SUMMARY S2). Reactors were cultivated under the sinLD cycle profile for an additional TR:TREATMENT_SUMMARY day of entrainment prior to sampling (total of 3 days of entrainment). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Briefly, 6 mL of 75% methanol (MeOH) was added to pellets, vortexed, and SP:SAMPLEPREP_SUMMARY transferred to glass vials. 9 mL of 100% methyl tert-butyl ether (MTBE) was SP:SAMPLEPREP_SUMMARY added, vortexed for 30 seconds, placed on automatic shaker for 1.5 hours at 4 SP:SAMPLEPREP_SUMMARY ºC, and sonicated for 15 minutes. 3.75 mL of water was added, each extraction SP:SAMPLEPREP_SUMMARY was vortexed by hand for 1 minute, and centrifuged for 10 minutes at 3,270g at SP:SAMPLEPREP_SUMMARY 4ºC. A biphasic solution with a pellet formed: the top, green MTBE layer and SP:SAMPLEPREP_SUMMARY the bottom, clear MeOH:H2O layer were separated into separate tubes and dried SP:SAMPLEPREP_SUMMARY under N2,gas overnight. The pellet was stored at -80 ºC. After drying, the MTBE SP:SAMPLEPREP_SUMMARY layer was resuspended in 100 uL 1:1 toluene:MeOH, transferred to a LC-MS vial SP:SAMPLEPREP_SUMMARY insert, and stored at -80C for <1 month prior to MS analysis. The MeOH:H2O layer SP:SAMPLEPREP_SUMMARY was resuspended in 1 mL of 1:1 H2O:MeOH, transferred to a 1.7 mL centrifuge tube SP:SAMPLEPREP_SUMMARY and spun at 15,000g for 2 minutes at 4 ºC. The supernatant was split into two SP:SAMPLEPREP_SUMMARY 465 µL aliquots—one for GCMS and one for LC(HILIC)MS—in glass vials and SP:SAMPLEPREP_SUMMARY dried under N2,gas. The protocol outlined above is suitable for filter-quenched SP:SAMPLEPREP_SUMMARY cyanobacteria samples and centrifuged cell pellets. The polar methanol/water SP:SAMPLEPREP_SUMMARY fraction resulting from the biphasic extraction was processed for analysis by SP:SAMPLEPREP_SUMMARY hydrophilic interaction liquid chromatography (HILIC) LC-MS. Dried samples were SP:SAMPLEPREP_SUMMARY resuspended in 100 µL 1:1 H2O:MeOH and 10 µL were aliquoted into a pooled QC SP:SAMPLEPREP_SUMMARY sample. Samples were stored at -80 ºC until analysis. The pooled QC sample was SP:SAMPLEPREP_SUMMARY mixed and aliquoted into twelve vials. A QC injection was run every tenth SP:SAMPLEPREP_SUMMARY injection. The dried polar fraction for analysis by GC-MS was stored at -80 ºC SP:SAMPLEPREP_SUMMARY until derivatization, immediately prior to MS analysis. Samples were derivatized SP:SAMPLEPREP_SUMMARY in 30 uL methoxyamine HCl and 30 uL MSTFA, as specified in the following SP:SAMPLEPREP_SUMMARY section. Ten microliters were removed from each sample to create a pooled QC SP:SAMPLEPREP_SUMMARY sample, mixed, and aliquoted into thirteen vials. A QC sample was run after SP:SAMPLEPREP_SUMMARY every sixth injection. The non-polar MTBE phase was processed for non-targeted SP:SAMPLEPREP_SUMMARY LC-MS analysis. Twenty microliters from each sample were pooled, mixed, and SP:SAMPLEPREP_SUMMARY aliquoted into thirteen pooled QC samples. QC injections were placed after every SP:SAMPLEPREP_SUMMARY sixth injection. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For non-targeted LC-MS experiments, two microliters of extract were injected CH:CHROMATOGRAPHY_SUMMARY onto a Waters Acquity UPLC system in discrete, randomized blocks with a pooled CH:CHROMATOGRAPHY_SUMMARY QC injection after every 6 sample injections and separated using a Waters CH:CHROMATOGRAPHY_SUMMARY Acquity UPLC CSH Phenyl Hexyl column (1.7 µM, 1.0 x 100 mm), using a gradient CH:CHROMATOGRAPHY_SUMMARY from solvent A (2mM ammonium hydroxide, 0.1% formic acid) to solvent B CH:CHROMATOGRAPHY_SUMMARY (Acetonitrile, 0.1% formic acid). Injections were made in 100% A, held at 100% A CH:CHROMATOGRAPHY_SUMMARY for 1 min, ramped to 98% B over 12 minutes, held at 98% B for 3 minutes, and CH:CHROMATOGRAPHY_SUMMARY then returned to starting conditions over 0.05 minutes and allowed to CH:CHROMATOGRAPHY_SUMMARY re-equilibrate for 3.95 minutes, with a 200 µL/min constant flow rate. The CH:CHROMATOGRAPHY_SUMMARY column and samples were held at 65 °C and 6 °C, respectively. The column CH:CHROMATOGRAPHY_SUMMARY eluent was infused into a Waters Xevo G2 Q-TOF-MS with an electrospray source in CH:CHROMATOGRAPHY_SUMMARY positive mode, scanning 50-2000 m/z at 0.2 seconds per scan, alternating between CH:CHROMATOGRAPHY_SUMMARY MS (6 V collision energy) and MSE mode (15-30 V ramp). Calibration was performed CH:CHROMATOGRAPHY_SUMMARY using sodium iodide with 1 ppm mass accuracy. The capillary voltage was held at CH:CHROMATOGRAPHY_SUMMARY 2200 V, source temp at 150 °C, and nitrogen desolvation temp at 350 °C with a CH:CHROMATOGRAPHY_SUMMARY flow rate of 800 L/hr. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Xevo G2 CH:COLUMN_NAME Waters Acquity UPLC CSH Phenyl Hexyl (1.7 uM, 1.0 x 100 mm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo QS MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Compounds were created by clustering features using RAMClustR (Broeckling et al. MS:MS_COMMENTS 2014). RAMClustR uses a similarity matric which calculates feature correlation MS:MS_COMMENTS across samples and retention time correlation between features. Hierarchical MS:MS_COMMENTS clustering of the similarity matrix was computed via the fastcluter package MS:MS_COMMENTS (Müllner 2013). The resulting clustered dendrogram is cut using DynamicTreeCut MS:MS_COMMENTS and spectra are created with clusters and features abundances from input data MS:MS_COMMENTS (Langfelder, Zhang, and Horvath 2008). The abundance for each mass in spectra is MS:MS_COMMENTS a weighted mean of feature intensity. The RAMClustR outputs are compounds MS:MS_COMMENTS (clusters of correlated features) and intensities for each sample; spectral MS:MS_COMMENTS abundance intensities reflect weighted mean of all features within the compound. MS:MS_RESULTS_FILE ST001199_AN001995_Results.txt UNITS:spectral abundance per cell Has m/z:No Has RT:No RT units:No RT data #END