#METABOLOMICS WORKBENCH wernerajz_20190302_123751_mwtab.txt DATATRACK_ID:1660 STUDY_ID:ST001197 ANALYSIS_ID:AN001993 PROJECT_ID:PR000808 VERSION 1 CREATED_ON June 18, 2019, 9:44 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 GC-MS Analysis of Insoluble/Polymeric Amino Acids (part-III) 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-5a-1 time:-2 SUBJECT_SAMPLE_FACTORS - 2-Synechocystis_6803-cell-5a-2 time:-2 SUBJECT_SAMPLE_FACTORS - 3-Synechocystis_6803-cell-5a-3 time:-2 SUBJECT_SAMPLE_FACTORS - 4-Synechocystis_6803-cell-6a-1 time:-1 SUBJECT_SAMPLE_FACTORS - 5-Synechocystis_6803-cell-6a-2 time:-1 SUBJECT_SAMPLE_FACTORS - 6-Synechocystis_6803-cell-6a-3 time:-1 SUBJECT_SAMPLE_FACTORS - 7-Synechocystis_6803-cell-630a-1 time:-0.5 SUBJECT_SAMPLE_FACTORS - 8-Synechocystis_6803-cell-630a-2 time:-0.5 SUBJECT_SAMPLE_FACTORS - 9-Synechocystis_6803-cell-630a-3 time:-0.5 SUBJECT_SAMPLE_FACTORS - 10-Synechocystis_6803-cell-7a-1 time:- SUBJECT_SAMPLE_FACTORS - 11-Synechocystis_6803-cell-7a-2 time:- SUBJECT_SAMPLE_FACTORS - 12-Synechocystis_6803-cell-7a-3 time:- SUBJECT_SAMPLE_FACTORS - 13-Synechocystis_6803-cell-730a-1 time:0.5 SUBJECT_SAMPLE_FACTORS - 14-Synechocystis_6803-cell-730a-2 time:0.5 SUBJECT_SAMPLE_FACTORS - 15-Synechocystis_6803-cell-730a-3 time:0.5 SUBJECT_SAMPLE_FACTORS - 16-Synechocystis_6803-cell-8a-1 time:1 SUBJECT_SAMPLE_FACTORS - 17-Synechocystis_6803-cell-8a-2 time:1 SUBJECT_SAMPLE_FACTORS - 18-Synechocystis_6803-cell-8a-3 time:1 SUBJECT_SAMPLE_FACTORS - 19-Synechocystis_6803-cell-9a-1 time:2 SUBJECT_SAMPLE_FACTORS - 20-Synechocystis_6803-cell-9a-2 time:2 SUBJECT_SAMPLE_FACTORS - 21-Synechocystis_6803-cell-9a-3 time:2 SUBJECT_SAMPLE_FACTORS - 22-Synechocystis_6803-cell-11a-1 time:4 SUBJECT_SAMPLE_FACTORS - 23-Synechocystis_6803-cell-11a-2 time:4 SUBJECT_SAMPLE_FACTORS - 24-Synechocystis_6803-cell-11a-3 time:4 SUBJECT_SAMPLE_FACTORS - 25-Synechocystis_6803-cell-1p-1 time:6 SUBJECT_SAMPLE_FACTORS - 26-Synechocystis_6803-cell-1p-2 time:6 SUBJECT_SAMPLE_FACTORS - 27-Synechocystis_6803-cell-1p-3 time:6 SUBJECT_SAMPLE_FACTORS - 28-Synechocystis_6803-cell-3p-1 time:8 SUBJECT_SAMPLE_FACTORS - 29-Synechocystis_6803-cell-3p-2 time:8 SUBJECT_SAMPLE_FACTORS - 30-Synechocystis_6803-cell-3p-3 time:8 SUBJECT_SAMPLE_FACTORS - 31-Synechocystis_6803-cell-5p-1 time:10 SUBJECT_SAMPLE_FACTORS - 32-Synechocystis_6803-cell-5p-2 time:10 SUBJECT_SAMPLE_FACTORS - 33-Synechocystis_6803-cell-5p-3 time:10 SUBJECT_SAMPLE_FACTORS - 34-Synechocystis_6803-cell-6p-1 time:11 SUBJECT_SAMPLE_FACTORS - 35-Synechocystis_6803-cell-6p-2 time:11 SUBJECT_SAMPLE_FACTORS - 36-Synechocystis_6803-cell-6p-3 time:11 SUBJECT_SAMPLE_FACTORS - 37-Synechocystis_6803-cell-630p-1 time:11.5 SUBJECT_SAMPLE_FACTORS - 38-Synechocystis_6803-cell-630p-2 time:11.5 SUBJECT_SAMPLE_FACTORS - 39-Synechocystis_6803-cell-630p-3 time:11.5 SUBJECT_SAMPLE_FACTORS - 40-Synechocystis_6803-cell-7p-1 time:12 SUBJECT_SAMPLE_FACTORS - 41-Synechocystis_6803-cell-7p-2 time:12 SUBJECT_SAMPLE_FACTORS - 42-Synechocystis_6803-cell-7p-3 time:12 SUBJECT_SAMPLE_FACTORS - 43-Synechocystis_6803-cell-730p-1 time:12.5 SUBJECT_SAMPLE_FACTORS - 44-Synechocystis_6803-cell-730p-2 time:12.5 SUBJECT_SAMPLE_FACTORS - 45-Synechocystis_6803-cell-730p-3 time:12.5 SUBJECT_SAMPLE_FACTORS - 46-Synechocystis_6803-cell-8p-1 time:13 SUBJECT_SAMPLE_FACTORS - 47-Synechocystis_6803-cell-8p-2 time:13 SUBJECT_SAMPLE_FACTORS - 48-Synechocystis_6803-cell-8p-3 time:13 SUBJECT_SAMPLE_FACTORS - 49-Synechocystis_6803-cell-9p-1 time:14 SUBJECT_SAMPLE_FACTORS - 50-Synechocystis_6803-cell-9p-2 time:14 SUBJECT_SAMPLE_FACTORS - 51-Synechocystis_6803-cell-9p-3 time:14 SUBJECT_SAMPLE_FACTORS - 52-Synechocystis_6803-cell-11p-1 time:16 SUBJECT_SAMPLE_FACTORS - 53-Synechocystis_6803-cell-11p-2 time:16 SUBJECT_SAMPLE_FACTORS - 54-Synechocystis_6803-cell-11p-3 time:16 SUBJECT_SAMPLE_FACTORS - 55-Synechocystis_6803-cell-1a-1 time:18 SUBJECT_SAMPLE_FACTORS - 56-Synechocystis_6803-cell-1a-2 time:18 SUBJECT_SAMPLE_FACTORS - 57-Synechocystis_6803-cell-1a-3 time:18 SUBJECT_SAMPLE_FACTORS - 58-Synechocystis_6803-cell-3a-1 time:20 SUBJECT_SAMPLE_FACTORS - 59-Synechocystis_6803-cell-3a-2 time:20 SUBJECT_SAMPLE_FACTORS - 60-Synechocystis_6803-cell-3a-3 time:20 SUBJECT_SAMPLE_FACTORS - 61-Synechocystis_6803-cell-5a_day2-1 time:22 SUBJECT_SAMPLE_FACTORS - 62-Synechocystis_6803-cell-5a_day2-2 time:22 SUBJECT_SAMPLE_FACTORS - 63-Synechocystis_6803-cell-5a_day2-3 time:22 SUBJECT_SAMPLE_FACTORS - 64-Synechocystis_6803-cell-6a_day2-1 time:23 SUBJECT_SAMPLE_FACTORS - 65-Synechocystis_6803-cell-6a_day3-2 time:23 SUBJECT_SAMPLE_FACTORS - 66-Synechocystis_6803-cell-6a_day4-3 time:23 SUBJECT_SAMPLE_FACTORS - 67-Synechocystis_6803-cell-630a_day2-1 time:23.5 SUBJECT_SAMPLE_FACTORS - 68-Synechocystis_6803-cell-630a_day3-2 time:23.5 SUBJECT_SAMPLE_FACTORS - 69-Synechocystis_6803-cell-630a_day4-3 time:23.5 SUBJECT_SAMPLE_FACTORS - 70-Synechocystis_6803-cell-7a_day2-1 time:24 SUBJECT_SAMPLE_FACTORS - 71-Synechocystis_6803-cell-7a_day2-2 time:24 SUBJECT_SAMPLE_FACTORS - 72-Synechocystis_6803-cell-7a_day2-3 time:24 SUBJECT_SAMPLE_FACTORS - QC-1-1 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-8 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-15 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-22 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-29 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-36 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-43 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-50 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-57 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-64 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-71 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-78 time:26 SUBJECT_SAMPLE_FACTORS - QC-1-85 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. An acid hydrolysis protocol was developed for identification of SP:SAMPLEPREP_SUMMARY amino acids, nucleosides, and carbohydrates bound in insoluble pellet of SP:SAMPLEPREP_SUMMARY protein, DNA/RNA, and polysaccharides, respectively. Pellets remaining from the SP:SAMPLEPREP_SUMMARY biphasic extraction were removed from storage at -80 degrees C and residual SP:SAMPLEPREP_SUMMARY solvent was evaporated under nitrogen gas. Pellets were re-suspended in 3 mL of SP:SAMPLEPREP_SUMMARY 6 M hydrochloric acid (HCl) using vigorous vortexing and pipette re-suspension. SP:SAMPLEPREP_SUMMARY The resulting suspension was a bright teal. The suspension was transferred SP:SAMPLEPREP_SUMMARY equally two three separate glass vials for hydrolysis of the separate polymer SP:SAMPLEPREP_SUMMARY constituents. Hydrolysis of proteins to amino acids was completed with a SP:SAMPLEPREP_SUMMARY hydrochloric acid (HCl) hydrolysis, based on previously published protocols SP:SAMPLEPREP_SUMMARY (Fountoulakis and Lahm 1998). Briefly, vials were incubated at 110 degrees C for SP:SAMPLEPREP_SUMMARY with a loose cap seal. After 4 hours, the acid in each vial was entirely SP:SAMPLEPREP_SUMMARY evaporated; 1 mL of 6 M HCl was added to each vial, vortexed, sealed tightly, SP:SAMPLEPREP_SUMMARY and returned to 110 degrees C. After a total of 24 hours, vials were removed, SP:SAMPLEPREP_SUMMARY and remaining acid was evaporated under nitrogen gas. Samples were resuspended SP:SAMPLEPREP_SUMMARY in 150 µL of 1:1 MeOH:H2O, 20 uL was removed to create a pooled QC sample, the SP:SAMPLEPREP_SUMMARY pooled QC was aliquoted into fourteen vials, and the solvent was evaporated SP:SAMPLEPREP_SUMMARY under nitrogen gas. Amino acid samples were derivatized in 30 uL of methoxyamine SP:SAMPLEPREP_SUMMARY HCl in pyridine and 30 uL of MTBSTFSA. The peak integration of each amino acid SP:SAMPLEPREP_SUMMARY in each sample was manually checked and curated in the software Chromeleon™. SP:SAMPLEPREP_SUMMARY Aspartic acid with 2- and 3-derivitization agent modifications were detected and SP:SAMPLEPREP_SUMMARY summed for the total spectral abundance. Threonine with 2- and 3-derivitization SP:SAMPLEPREP_SUMMARY agent modifications were detected and summed for the total spectral abundance. SP:SAMPLEPREP_SUMMARY Polysaccharides and nucleic acid polymers were hydrolyzed to nucleosides using a SP:SAMPLEPREP_SUMMARY modified protocol from Huang et al. (2012) (Huang, Kaiser, and Benner 2012). SP:SAMPLEPREP_SUMMARY Briefly, vials were incubated at 130 degrees C for 10 minutes, removed and SP:SAMPLEPREP_SUMMARY allowed to cool, and 100 uL was transferred to a glass teardrop vial. The SP:SAMPLEPREP_SUMMARY remaining pellet was incubated at 160 degrees C for 40 minutes, removed and SP:SAMPLEPREP_SUMMARY allowed to cool, re-suspended in 100 uL LC-MS grade water, vortexed for 15 SP:SAMPLEPREP_SUMMARY seconds, centrifuged at 1,500g for 2 minutes, and the supernatant was SP:SAMPLEPREP_SUMMARY transferred to the glass teardrop vial which contained purines from the 130 SP:SAMPLEPREP_SUMMARY degree C incubation. The acid was evaporated under nitrogen gas. Samples were SP:SAMPLEPREP_SUMMARY removed after the 10 minute 130ºC incubation to preserve purines (guanine and SP:SAMPLEPREP_SUMMARY adenine) from degradation during the 40 minute 160ºC incubation. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For non-targeted GC-MS experiments, metabolites were detected using a Trace 1310 CH:CHROMATOGRAPHY_SUMMARY GC coupled to a Thermo ISQ mass spectrometer. Samples (1 µL) were injected at a CH:CHROMATOGRAPHY_SUMMARY 10:1 split ratio to a 30 m TG-5MS column (Thermo Scientific, 0.25 mm i.d., 0.25 CH:CHROMATOGRAPHY_SUMMARY μm film thickness) with a 1.2 mL/min helium gas flow rate. GC inlet was held at CH:CHROMATOGRAPHY_SUMMARY 285°C. The oven program started at 140°C for 1 min, followed by a ramp of CH:CHROMATOGRAPHY_SUMMARY 15°C/min to 330°C, and 5 min hold. Masses between 50-650 m/z were scanned at 5 CH:CHROMATOGRAPHY_SUMMARY scans/sec under electron impact ionization. Transfer line and ion source were CH:CHROMATOGRAPHY_SUMMARY held at 300 and 260°C, respectively. Pooled QC samples were injected after CH:CHROMATOGRAPHY_SUMMARY every 6 actual samples. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Thermo ISQ CH:COLUMN_NAME Trace 1310 GC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo ISQ MS:INSTRUMENT_TYPE GC-TOF MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Raw data was converted to *.CSV with Waters® Databridge. For idMS/MS (RP-LC-MS MS:MS_COMMENTS runs), a file was converted for low-collision, high-collision, and LockSpray for MS:MS_COMMENTS each sample. Peaks were detected within the XCMS workflow using the Centwave MS:MS_COMMENTS algorithm (Smith et al. 2006). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS spectral abundance per cell MS_METABOLITE_DATA_START Samples 1-Synechocystis_6803-cell-5a-1 2-Synechocystis_6803-cell-5a-2 3-Synechocystis_6803-cell-5a-3 4-Synechocystis_6803-cell-6a-1 5-Synechocystis_6803-cell-6a-2 6-Synechocystis_6803-cell-6a-3 7-Synechocystis_6803-cell-630a-1 8-Synechocystis_6803-cell-630a-2 9-Synechocystis_6803-cell-630a-3 10-Synechocystis_6803-cell-7a-1 11-Synechocystis_6803-cell-7a-2 12-Synechocystis_6803-cell-7a-3 13-Synechocystis_6803-cell-730a-1 14-Synechocystis_6803-cell-730a-2 15-Synechocystis_6803-cell-730a-3 16-Synechocystis_6803-cell-8a-1 17-Synechocystis_6803-cell-8a-2 18-Synechocystis_6803-cell-8a-3 19-Synechocystis_6803-cell-9a-1 20-Synechocystis_6803-cell-9a-2 21-Synechocystis_6803-cell-9a-3 22-Synechocystis_6803-cell-11a-1 23-Synechocystis_6803-cell-11a-2 24-Synechocystis_6803-cell-11a-3 25-Synechocystis_6803-cell-1p-1 26-Synechocystis_6803-cell-1p-2 27-Synechocystis_6803-cell-1p-3 28-Synechocystis_6803-cell-3p-1 29-Synechocystis_6803-cell-3p-2 30-Synechocystis_6803-cell-3p-3 31-Synechocystis_6803-cell-5p-1 32-Synechocystis_6803-cell-5p-2 33-Synechocystis_6803-cell-5p-3 34-Synechocystis_6803-cell-6p-1 35-Synechocystis_6803-cell-6p-2 36-Synechocystis_6803-cell-6p-3 37-Synechocystis_6803-cell-630p-1 38-Synechocystis_6803-cell-630p-2 39-Synechocystis_6803-cell-630p-3 40-Synechocystis_6803-cell-7p-1 41-Synechocystis_6803-cell-7p-2 42-Synechocystis_6803-cell-7p-3 43-Synechocystis_6803-cell-730p-1 44-Synechocystis_6803-cell-730p-2 45-Synechocystis_6803-cell-730p-3 46-Synechocystis_6803-cell-8p-1 47-Synechocystis_6803-cell-8p-2 48-Synechocystis_6803-cell-8p-3 49-Synechocystis_6803-cell-9p-1 50-Synechocystis_6803-cell-9p-2 51-Synechocystis_6803-cell-9p-3 52-Synechocystis_6803-cell-11p-1 53-Synechocystis_6803-cell-11p-2 54-Synechocystis_6803-cell-11p-3 55-Synechocystis_6803-cell-1a-1 56-Synechocystis_6803-cell-1a-2 57-Synechocystis_6803-cell-1a-3 58-Synechocystis_6803-cell-3a-1 59-Synechocystis_6803-cell-3a-2 60-Synechocystis_6803-cell-3a-3 61-Synechocystis_6803-cell-5a_day2-1 62-Synechocystis_6803-cell-5a_day2-2 63-Synechocystis_6803-cell-5a_day2-3 64-Synechocystis_6803-cell-6a_day2-1 65-Synechocystis_6803-cell-6a_day3-2 66-Synechocystis_6803-cell-6a_day4-3 67-Synechocystis_6803-cell-630a_day2-1 68-Synechocystis_6803-cell-630a_day3-2 69-Synechocystis_6803-cell-630a_day4-3 70-Synechocystis_6803-cell-7a_day2-1 71-Synechocystis_6803-cell-7a_day2-2 72-Synechocystis_6803-cell-7a_day2-3 QC-1-1 QC-1-8 QC-1-15 QC-1-22 QC-1-29 QC-1-36 QC-1-43 QC-1-50 QC-1-57 QC-1-64 QC-1-71 QC-1-78 QC-1-85 Factors time:-2 time:-2 time:-2 time:-1 time:-1 time:-1 time:-0.5 time:-0.5 time:-0.5 time:- time:- time:- time:0.5 time:0.5 time:0.5 time:1 time:1 time:1 time:2 time:2 time:2 time:4 time:4 time:4 time:6 time:6 time:6 time:8 time:8 time:8 time:10 time:10 time:10 time:11 time:11 time:11 time:11.5 time:11.5 time:11.5 time:12 time:12 time:12 time:12.5 time:12.5 time:12.5 time:13 time:13 time:13 time:14 time:14 time:14 time:16 time:16 time:16 time:18 time:18 time:18 time:20 time:20 time:20 time:22 time:22 time:22 time:23 time:23 time:23 time:23.5 time:23.5 time:23.5 time:24 time:24 time:24 time:26 time:26 time:26 time:26 time:26 time:26 time:26 time:26 time:26 time:26 time:26 time:26 time:26 alanine 1898550368 1693817107 1702092432 2001255008 1696525746 1938079377 1663203741 1827730000 1744216171 1779795650 1996864066 1468671007 1549437872 1794363905 2089033265 1555309688 1878825654 1929900127 1987162674 1818567732 1817064023 1808879607 2206581493 1830902552 1964241906 1664613693 2281873270 1838043253 1643621218 1547212977 1367403264 1378220328 1412010985 1461776434 1421235083 1227779038 1171515862 1083692719 1242415827 1039157895 1144086343 1047394184 1073116695 1127126379 1258384490 1088498657 1155313705 1234128564 1225406302 1583504735 1215775854 1138707352 1321422686 1124880514 1303222132 1179010926 1284747918 1114528316 1224632008 1069429951 1244969573 1213570814 1033051515 1191653867 779565475.4 900034571.4 1110005891 1235535515 1423695805 1650163075 1011077937 1146051751 852521795.9 716460714.3 740304204.1 752462051 773760785.7 753527530.6 772914811.2 792915760.2 784018627.6 793619515.3 795902255.1 782648413.3 794054607.1 Glycine 989079486.1 831134641.2 844618416.7 1088906177 914538612.7 963079852.5 811103870.7 972801185.2 861600902.4 885220139.9 977155090.2 719714881.6 777275113.5 823944664.2 1035873508 404174432.6 852447007.5 1014136716 1117061080 1028409634 1020607267 972714550 1290528813 965895944.1 1101441187 960191784.3 1267296643 1019752353 965505866.7 855883789.5 750164383.4 807670429.3 747765613.9 874747402.1 718426936.3 715627636.4 637254085.4 571223242.6 728381256.6 592065707.6 581602457.6 554252643.4 544582979.1 621257540.2 621242753.1 387381585.9 517105366.5 512178284 487044240 705525122.4 331126648.1 201697652.8 607879924.7 605660506 542707478.1 552438311.5 603468434.8 397310109.4 498414301.9 505971851.6 663095950.2 566257310.1 491770127.7 574276907.7 403069259 474429416.1 632955899.2 635964528.6 861810307 835727714.3 559976505.9 601931108 460378382.7 348002622.4 346097020.4 357696352 385031581.6 375122306.1 361370739.8 370044357.1 371355107.1 371212928.6 372537061.2 387761775.5 360431964.3 X2 aminobutyric acid 15291805.56 25324572.52 26091553.03 8127138.462 5701126.761 22276877.05 26106489.8 10177762.96 30521439.02 30055104.9 19908040.98 28816263.16 29317773.05 41062430.66 34542568.18 124796326.2 45562977.44 32168201.49 8399123.188 6855415.493 4256152.672 14045678.57 7015037.313 11408265.73 4674302.158 5778620.915 12304230.16 11491560 5347793.939 3515736.842 10953212.44 3977388.889 11477910.89 3838587.302 21562401.96 2762961.722 12390479.67 6648889.362 5629181.416 4857332.13 14690851.69 15831676.23 14828828.45 6147318.008 21432522.63 67281585.86 28827059.76 63608703.7 52568128.89 64761596.94 132120596.6 82001638.89 43413104.6 13288152.61 35485434.21 29257102.46 23483057.97 80055699.22 77735343.4 33157240.28 24497419.09 42524996.12 22822489.05 27957966.79 3420213.115 11884602.48 4306991.935 21374726.87 5336139.535 21776937.89 3204996.078 2254309.859 15698836.73 12136454.08 12478229.59 12813010.2 13324627.55 13054336.73 12755551.02 12634545.92 12639704.08 12063780.61 11984051.02 12151867.35 11945632.65 valine 1088666910 981814717.6 904364090.9 1142711062 980979767.6 1048519885 887769088.4 1076368089 921958130.1 966890874.1 1152922541 834127289.5 875040177.3 987888496.4 1214200545 460092503.5 1044808316 1138635530 1277925891 1238895134 1154311901 1100786650 1427866410 1027698706 1228148295 1056819987 1342599944 1105065227 1054473073 949683578.9 892204730.6 966142500 937886089.1 1028915587 871683892.2 850139717.7 822100991.9 700523591.5 835490601.8 706481108.3 678135000 606652409.8 630766255.2 741768685.8 800917378.6 557276026.9 687818924.3 707792119.3 647199448.9 979787892.9 439443888.4 371099245.4 799238213.4 737101232.9 735903223.7 691529832 772546830.9 509067214.8 636559449.1 650224632.5 785323966.8 705289569.8 602510649.6 722082468.6 481767855.7 542905130.4 697300560.5 678273770.9 967012316.3 927256099.4 639956176.5 655916887.3 467381801 387580270.4 399471112.2 406732234.7 422259357.1 410580913.3 412651367.3 423278586.7 414624204.1 421158566.3 420994494.9 420872357.1 419594545.9 leucine 1103963375 1022052908 1043148689 1168554354 1075705493 1159144344 945734551 1165936667 1026456862 1023646462 1160624049 847801230.3 896090787.2 963142627.7 1192781152 612908851.1 1050363880 1171955940 1283592109 1138810859 1115298962 1088462436 1602497918 1145411860 1303604460 1138152359 1397728032 1191364013 1172563879 1046086620 947239943 950810893.9 944361044.6 1026846259 919282392.2 834471813.4 769472780.5 687496783 866019637.2 709950259.9 738310008.5 628643713.1 692599594.1 756540988.5 758750086.4 620679771 669304976.1 726132382.7 677505022.2 1018489653 563240467.8 451362731.5 813858506.3 728560429.7 760574201.8 685686692.6 743146628 580874019.5 701532566 669283371 777495800.8 753466802.3 628915397.8 749459826.6 494936639.3 589168304.3 730170056.5 760100986.8 995439181.4 980208087 690889011.8 743083943.7 457273454.1 398776561.2 412113699 415802280.6 434080244.9 423295285.7 429711872.4 435852178.6 434536224.5 429103137.8 438384326.5 436577433.7 430042290.8 isoleucine 732937006.9 672511618.3 607082590.9 770809100 675890549.3 704191475.4 599633278.9 724524244.4 615907170.7 668134090.9 762086549.2 564429375 578350652.5 658262605.8 855115287.9 286290134.8 708988827.1 783538634.3 927900971 849963323.9 802310893.1 750022021.4 1071299463 700625902.1 874441410.1 769748143.8 954797309.5 769466500 754335048.5 676394122.8 655721041.5 681589338.4 669371618.8 746446232.8 621997975.5 614374535.9 605898638.2 494364425.5 622037367.3 509451953.1 486678360.2 417199979.5 450422636 540067383.1 575838407.4 121099437.7 483810533.9 514449230.5 449347302.2 723610357.1 294465158.8 232759944.4 578539230.1 540434614.5 526497004.4 488790881.1 523330391.3 342008605.5 457530426.4 475844678.4 561438925.3 510813748.1 426696237.2 536303070.1 327419675.4 392477922.4 501267935.5 474875947.1 704141018.6 641515689.4 458204568.6 463215009.4 303472158.2 260545596.9 266606459.2 274454066.3 282844234.7 276787316.3 276537658.2 283607494.9 281540811.2 280596826.5 284291301 281376178.6 280775535.7 Threonine_R1 27086763.89 26480572.52 40816386.36 36642061.54 39154000 38976713.11 29757870.75 33942518.52 39800821.14 24025482.52 19890270.49 25659986.84 29521049.65 15508970.8 18418825.76 14323730.5 25553601.5 38301328.36 25473101.45 17334683.1 27253793.89 22316050 57956753.73 38715377.62 31361230.22 43142921.57 37192928.57 39463560 42166036.36 28636146.2 28071010.36 22105141.41 16457470.3 29268486.77 15832818.63 13882220.1 8605414.634 16287710.64 23901464.6 15535267.15 22921419.49 21531372.95 27021326.36 14645624.52 13924617.28 8161491.582 8369601.594 6786345.679 11955626.67 14822806.12 7752017.167 11886171.3 12794786.61 13277598.39 9994671.053 16841663.93 13088289.86 7631566.406 12084554.72 9643385.159 17830186.72 14553337.21 14629569.34 8512387.454 10690750.82 20113065.22 19927842.74 25550488.99 36927381.4 22306521.74 26152117.65 18926173.71 34556209.18 24979280.61 26523127.55 26385663.27 27998346.94 26123933.67 26317367.35 26123897.96 26044627.55 25392984.69 24728887.76 24851872.45 21087107.14 Proline 496734006.9 392322961.8 401694128.8 503508192.3 424016535.2 454421139.3 382330551 461803437 378884032.5 410003524.5 470214008.2 319791078.9 329125354.6 389183788.3 497532075.8 323776269.5 404170210.5 440832567.2 534779289.9 496191577.5 471388160.3 446805500 644250268.7 466374734.3 551148223 447495339.9 590594134.9 477572926.7 455761678.8 417112292.4 380234777.2 384229126.3 374332861.4 421751920.6 360774696.1 334656052.6 338362455.3 264423638.3 333633460.2 278722465.7 274493809.3 242852815.6 255193481.2 307440613 309821732.5 269506596 276038581.7 304680683.1 270956577.8 414006413.3 263123017.2 249734699.1 316715129.7 303044847.4 329170425.4 280783192.6 309900067.6 255913097.7 281106264.2 268316844.5 306035074.7 294153391.5 239805324.8 313650557.2 183113934.4 225371344.7 286594806.5 288782859 401124186 387663764 257693537.3 282370544.6 168459821.4 142376699 146167765.3 151282658.2 155167831.6 149288974.5 151302566.3 155083454.1 153575770.4 153638785.7 153548479.6 153164693.9 152562443.9 aspartic acid_R1 36174236.11 45402061.07 39966818.18 47609338.46 28214260.56 50184500 39746081.63 47431162.96 39385455.28 41790258.74 49927106.56 34674315.79 32957496.45 49202510.95 57440000 10173191.49 36058060.15 43454447.76 53642028.99 68793394.37 58176725.19 49132178.57 110341223.9 56735258.74 70321820.14 51772692.81 70330666.67 60273340 66264963.64 51078438.6 54493849.74 38044429.29 42174490.1 45491301.59 42056769.61 42575569.38 35163731.71 33766153.19 35619787.61 33091158.84 33325991.53 23070651.64 25901820.08 38789697.32 32016074.07 15316377.1 30362370.52 23016539.09 22438257.78 51213392.86 9032811.159 6657254.63 40142435.15 38245751 34204868.42 24369422.13 24509512.08 18278464.84 17602905.66 26048565.37 28767406.64 28878108.53 28023715.33 40033896.68 20935137.7 19810571.43 30882395.16 31955541.85 56471027.91 38418838.51 33823850.98 26654896.71 9893377.551 12375045.92 9179443.878 16538520.41 15620061.22 21429479.59 19307545.92 14210928.57 16814938.78 19526045.92 19731229.59 15021867.35 14739867.35 Glutamine pyroglutamic acid 226945256.9 250324603.1 173665697 206061284.6 104202331 129851918 108633693.9 186638059.3 202198609.8 131352615.4 379502254.1 285031572.4 46857304.96 540151839.4 276041621.2 863116312.1 541309864.7 256678067.2 189744869.6 347607570.4 146754175.6 348363571.4 260354238.8 205729685.3 236522812.9 211510483.7 269606055.6 223246560 209052781.8 184020479.5 310678538.9 165220853.5 417892920.8 162530089.9 433099818.6 122957655.5 415298995.9 244130897.9 208663973.5 153424447.7 141365084.7 64960500 54111435.15 191880164.8 347796950.6 594944387.2 484776354.6 621260135.8 482481413.3 741849663.3 721426888.4 662318828.7 552216991.6 321529228.9 640309184.2 458588582 488225748.8 592458019.5 556505120.8 479933494.7 452392643.2 490413922.5 362840562 489393546.1 160771737.7 98344378.88 122638621 138494096.9 178670841.9 150837795 116179494.1 129718802.8 171482290.8 154320035.7 160604464.3 161936484.7 161968607.1 153478000 158644622.4 164116913.3 159823056.1 158755020.4 160845398 157335714.3 160401474.5 Methionine 131743680.6 216435259.5 111289045.5 148339569.2 134249133.8 106469393.4 82735278.91 149810807.4 101074609.8 124032643.4 171211041 127324296.1 144363943.3 197722795.6 220744575.8 95841070.92 214039827.1 246137343.3 316085108.7 318269387.3 324680412.2 252298778.6 276887037.3 135957524.5 166416877.7 183928516.3 300558611.1 130692300 223609097 119498386 251431285 224183479.8 227725940.6 298551899.5 175226642.2 235292578.9 214284337.4 133565834 187737800.9 707385288.8 79576597.46 85203778.69 79430661.09 120300785.4 120731510.3 121106175.1 128113139.4 108285827.2 111517168.9 228961408.2 79907656.65 64260800.93 166953799.2 198846550.2 175302872.8 141784860.7 192445154.6 102894418 124163584.9 161313056.5 185027771.8 143079023.3 144209339.4 176043841.3 115402662.3 132244698.8 117780996 110834577.1 183221586 129520453.4 180915415.7 180559910.8 88981464.29 79785387.76 84248255.1 83734591.84 84221479.59 78780219.39 78329096.94 81044397.96 78679051.02 76986831.63 77419255.1 74464341.84 75290448.98 serine 1160022542 878085572.5 938466189.4 1261200362 1136810746 1111369451 852511564.6 1086784133 849851504.1 875588055.9 840134975.4 605232585.5 686622865.2 454741941.6 933328075.8 72819304.96 535918255.6 942034783.6 1231195188 1081980796 1171219466 1004944650 1758762769 1226107692 1499322799 1235230157 1629355857 1313868447 1284154958 1137861269 819142513 1035651848 746332386.1 1091036376 566442857.8 869533612.4 584737296.7 547644851.1 839488115 391417018.1 677419466.1 569462000 148737535.6 759095946.4 541945872.4 100080723.9 241857004 156984172.8 172313760 277469668.4 34640549.36 72402875 277450276.2 539854662.7 246608342.1 290969209 357713304.3 85414316.41 142695343.4 223629735 403640199.2 256309794.6 274965200.7 323839302.6 402852862.3 478740673.9 736943939.5 637958207 1097051200 906316273.3 695187721.6 770749042.3 305299760.2 298599433.7 305190290.8 310969489.8 314870933.7 312308846.9 319404709.2 325269581.6 320306321.4 325040750 323665229.6 318323653.1 323848204.1 Threonine_R2 668011020.8 385776183.2 289989515.2 569862523.1 418812478.9 317772639.3 386077863.9 562470748.1 199226260.2 477871839.2 536061737.7 185348473.7 208056177.3 360721007.3 569820348.5 41084588.65 189060639.1 333101044.8 700917478.3 643490802.8 637569801.5 507771821.4 791405313.4 551620678.3 915174892.1 509655359.5 805382976.2 579157426.7 494949200 600087251.5 316958569.9 616451904 518623638.6 632564021.2 434256848 588896081.3 450742674.8 191226234 395244376.1 543067924.2 221512817.8 219269057.4 160873585.8 499828773.9 325108049.4 150178111.1 269696892.4 232088119.3 99739022.22 248648377.6 87845927.04 40353439.81 208387050.2 262846610.4 244424456.1 166865991.8 239738014.5 86812804.69 145168581.1 239996250.9 259385336.1 214404449.6 146647689.8 328278439.1 280933078.7 220711987.6 433059391.1 282768052.9 550120683.7 518102173.9 239537329.4 501203488.3 34416091.84 36144627.55 43938066.33 50355301.02 57476653.06 62501561.22 73543459.18 81896709.18 86739219.39 95748836.73 99290479.59 100483459.2 132148994.9 Phenylalanine 886003847.2 759278297.7 720888734.8 897889538.5 797296014.1 828633893.4 707181904.8 855833777.8 712831382.1 771260258.7 880582614.8 626302105.3 659647404.3 740392277.4 935995681.8 356521808.5 782234985 861039119.4 1011399471 927531239.4 878057687 848878000 1162963201 837788363.6 1004537669 837145875.8 1108551770 877911840 858683878.8 763575233.9 706875927.5 724741186.9 731234306.9 806534179.9 670186529.4 659854358.9 637521695.1 520437357.4 658159216.8 1220100744 547358004.2 468828885.2 502986715.5 581851003.8 611751284 421859562.3 497872330.7 531485037 464465217.8 748635898 318532540.8 239606527.8 609978405.9 572602124.5 565587622.8 512459155.7 579129430 388620605.5 475106188.7 495245091.9 586807145.2 530546949.6 447207262.8 568759988.9 359023327.9 433413146 558109653.2 540914251.1 770943925.6 715628142.9 500821800 525510436.6 305884602 291261913.3 298820224.5 305312346.9 315296596.9 302884117.3 306913311.2 318791949 313214346.9 313744418.4 311827668.4 308197653.1 307067040.8 aspartic acid_R2 2140980313 1849574084 1852035212 2208792331 2011810268 2138853148 1735409544 2123438437 1800492732 1822982196 2104255131 378628881.6 1566765986 1602635745 2148458417 154498687.9 1832598105 2065291627 2457862080 2303589254 469511167.9 2173368700 2994782537 2334598126 2553569719 2150909928 2750133373 2167452773 2066090624 1838528567 1641298358 1705344783 1567168767 1824383825 1486873598 1495177512 1376068602 1180152345 1469044788 1070285949 1227649000 1066032352 1141519063 1314898169 1274625502 788971316.5 1035190932 957932312.8 935574782.2 1449884827 337972922.7 87453041.67 1183693971 1242701301 1082084325 167313709 1217606643 619697203.1 883474071.7 952816137.8 1217687834 1043645554 957099642.3 1127461181 808967360.7 948761801.2 1229982157 1222143137 1691224195 1644888193 1150068910 1248227737 604628724.5 655745607.1 678608132.7 700168632.7 712705091.8 695237520.4 705967346.9 724576576.5 721578321.4 721359403.1 723747556.1 710046000 715528112.2 glutamic acid 1776348333 1611274435 1521564386 1764236531 1693978979 1601631172 1469222265 1633161533 1533268179 1658210497 1674288828 1168613697 1435337404 1223504701 1728283758 35031255.32 1291463586 1729630761 2008405000 1645153521 1693114863 1435437521 2474746037 1897680818 2168736252 1829707967 2164671421 1862723567 1763772879 1663132427 1344191627 1542834924 1132563356 1592514825 1098869397 1320352033 994645451.2 944929774.5 1281483978 586784436.8 1106843678 970588590.2 1019925703 1155332670 940842185.2 456428030.3 715472099.6 653878802.5 623271244.4 974496209.2 160527630.9 37445828.7 829500585.8 905317325.3 722269728.1 712235561.5 848970096.6 387122980.5 575042649.1 689541162.5 861968410.8 672383178.3 686474073 787924516.6 664243786.9 838747860.2 1067628722 1100693480 1420463614 1445957671 1036225675 1019820596 495446739.8 527258076.5 516772250 546618806.1 550326377.6 543668607.1 562296142.9 565307015.3 562714234.7 575140846.9 572189913.3 553373775.5 565739331.6 lysine 972749006.9 796352839.7 772020128.8 976536338.5 866908915.5 902379959 735893523.8 915949192.6 745143243.9 813465657.3 933365123 656874500 674309432.6 776041510.9 972007984.8 442629127.7 846572894.7 925884955.2 1082610681 991906584.5 940049969.5 917740635.7 1315401493 920431762.2 1147777403 942560588.2 1206128317 999881326.7 974990527.3 855112982.5 785835202.1 817157813.1 800904990.1 862763148.1 735075205.9 698414483.3 680870280.5 567795004.3 733889579.6 316769942.2 583714444.9 492529262.3 536923535.6 654362605.4 660534987.7 438888454.5 536584928.3 576913411.5 503151720 800078668.4 375991218.9 302061092.6 658071502.1 631993502 586280179.8 552360295.1 616594666.7 434684105.5 531255339.6 515927947 638973597.5 577747112.4 491113573 613085287.8 375806914.8 474602863.4 605057616.9 595633132.2 865026702.3 766312279.5 554259592.2 569135075.1 286947413.3 286817551 299732484.7 310242627.6 315426729.6 307530668.4 314636413.3 320306408.2 319411474.5 319974153.1 321042959.2 311460816.3 316523464.3 arginine 566247597.2 423323374 448655795.5 567886946.2 538852676.1 529614606.6 390010068 475865259.3 411546674.8 406753447.6 491364147.5 357542473.7 351793971.6 419190518.2 528921734.8 268219808.5 477230248.1 487400858.2 652816731.9 652577098.6 654272351.1 578410428.6 1037772978 651711706.3 704439518 672081339.9 746765468.3 516996420 619517193.9 454122309.9 384362020.7 458603298 373322524.8 486039756.6 338142014.7 377565933 360205215.4 299001319.1 387260632.7 278352436.8 311547881.4 252355172.1 295043895.4 309324992.3 309190148.1 241608686.9 257331394.4 263773625.5 286234613.3 434459729.6 175402017.2 155002222.2 308960004.2 324718325.3 310587377.2 260580573.8 314466512.1 232611668 262079826.4 242341000 295301717.8 250934317.8 229654109.5 274713878.2 175559144.3 218201304.3 287910778.2 274223330.4 494001316.3 375736664.6 311577478.4 285914441.3 153559035.7 142303025.5 145147811.2 147759918.4 133468127.6 133463290.8 143943071.4 143209255.1 140023219.4 142222867.3 142266780.6 132030438.8 140011239.8 histidine 451223291.7 361352320.6 374837947 468894446.2 424380711.3 439829541 357627292.5 422203570.4 363487918.7 382478895.1 432097647.5 316352348.7 329553404.3 357792664.2 466021151.5 139796425.5 401658458.6 445564835.8 509576007.2 486319373.2 454479771 426718100 616347709 426964042 536489611.5 444732006.5 572782436.5 461382666.7 459816436.4 400796005.8 372365896.4 377409020.2 354659594.1 415917201.1 326809093.1 340937636.4 310663048.8 271498761.7 334982181.4 564301292.4 275732792.4 232926577.9 251597920.5 290216318 289655580.2 180551831.6 228821302.8 228375567.9 212431222.2 350152178.6 112824171.7 104934120.4 278317284.5 285752959.8 263260258.8 243945606.6 281498246.4 158720222.7 204089901.9 228712586.6 282537734.4 247925562 226997146 265711564.6 181309957.4 220880841.6 278106000 282089559.5 400824130.2 359414329.2 266200611.8 263485647.9 119141556.1 125717688.8 130924489.8 134025081.6 134198591.8 129071954.1 129120520.4 133570443.9 131952091.8 132276071.4 132533852 128953295.9 129972408.2 tyrosine 952755291.7 835998648.9 828332947 1000267523 887801929.6 949122163.9 772242591.8 941293837 834600268.3 856242860.1 871165368.9 674521342.1 743570503.5 774790540.1 995564750 454563418.4 823509789.5 944871149.3 1085972413 1068144711 1037548710 908745600 1336192985 925719153.8 1172067993 989484052.3 1236631937 941662193.3 969539818.2 819861713.5 787918196.9 883562075.8 728836846.5 954535719.6 623447009.8 797698014.4 705716272.4 522666268.1 679361690.3 35344494.58 555179135.6 498744832 522065142.3 568726279.7 575601642 427956084.2 481029442.2 493481448.6 457700760 805309295.9 368990223.2 345147287 577541071.1 624351289.2 625167131.6 524812209 641822434.8 395293535.2 456682184.9 512897187.3 625589273.9 523317759.7 484480941.6 555665391.1 417550803.3 469190018.6 632457076.6 608730955.9 895681204.7 820565354 597401117.6 652185516.4 338261265.3 343730061.2 350017086.7 361976949 367942168.4 356807877.6 365904301 377223362.2 374473056.1 374959836.7 374804846.9 368122301 375446454.1 cystine 31269986.11 43497328.24 28865128.79 44624946.15 34048063.38 40489000 24093163.27 32785896.3 28005528.46 30340090.91 34279508.2 27563197.37 29876652.48 18880824.82 48247515.15 27458766.92 59245686.57 77655391.3 95785964.79 77315793.89 58375764.29 117915164.2 58180986.01 84301496.4 97703261.44 78011865.08 41110633.33 60052121.21 38917777.78 60777269.43 94322171.72 25158173.27 96119825.4 23676338.24 70511200.96 32659333.33 24857459.57 39617314.16 18472169.49 17742405.74 17850330.54 28092302.68 22578045.27 3917781.145 10199657.37 8862201.646 6059573.333 14574857.14 8063347.28 32587947.79 10127210.53 12736877.05 29508560.39 5221166.038 8493607.774 17554381.74 9503748.062 9623645.985 11583202.95 23912898.36 34987844.72 34959959.68 24535348.02 66495781.4 41682329.19 63722207.84 58407920.19 8167331.633 8986510.204 9590755.102 9443612.245 9640841.837 9669928.571 9064147.959 8923331.633 9111229.592 9235071.429 9759617.347 9327081.633 9513229.592 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name alanine Glycine X2 aminobutyric acid valine leucine isoleucine Threonine_R1 Proline aspartic acid_R1 Glutamine pyroglutamic acid Methionine serine Threonine_R2 Phenylalanine aspartic acid_R2 glutamic acid lysine arginine histidine tyrosine cystine METABOLITES_END #END