#METABOLOMICS WORKBENCH ManoelSouza_20230403_061719 DATATRACK_ID:3836 STUDY_ID:ST002537 ANALYSIS_ID:AN004176 PROJECT_ID:PR001633 VERSION 1 CREATED_ON April 3, 2023, 10:11 am #PROJECT PR:PROJECT_TITLE Osmoprotectants play a major role in the Portulaca oleracea resistance to high PR:PROJECT_TITLE levels of salinity stress - Insights from a metabolomics and proteomics PR:PROJECT_TITLE integrated approach PR:PROJECT_SUMMARY Purslane is an invasive plant and is considered the eighth most common weed in PR:PROJECT_SUMMARY the world. Because of that, its outdoor production in extensive areas faces PR:PROJECT_SUMMARY several concerns. Kong & Zheng (2014) evaluated the potential of producing PR:PROJECT_SUMMARY purslane in a hydroponic system, generating approximately 5.75 kg of fresh PR:PROJECT_SUMMARY matter per m2 per month, which might yield 57.5 tons/hectare/year if cultivated PR:PROJECT_SUMMARY in a bimestrial regime. The high productivity of purslane, when grown in PR:PROJECT_SUMMARY controlled-environment agriculture, can open many opportunities for the purslane PR:PROJECT_SUMMARY industry, even in the context of biosaline agriculture. Building up a robust PR:PROJECT_SUMMARY multi-omics database on the response of purslane to salt stress and the PR:PROJECT_SUMMARY subsequent study of it via an MOI analysis can create the basis for a future PR:PROJECT_SUMMARY system biology approach to decode the genetics behind its resistance to salinity PR:PROJECT_SUMMARY stress. The present study is a second step in building a robust database on the PR:PROJECT_SUMMARY morpho-physiological and molecular responses of Portulaca oleracea L. to PR:PROJECT_SUMMARY salinity stress and its subsequent use in attempting to decode the genetics PR:PROJECT_SUMMARY behind its resistance to this abiotic stress. After reporting on the PR:PROJECT_SUMMARY characterization of the morpho-physiological responses of young purslane plants PR:PROJECT_SUMMARY to such stress using a robust salinization protocol, here we report a study on PR:PROJECT_SUMMARY adult purslane plants through the characterization of the untargeted metabolome PR:PROJECT_SUMMARY and proteome profiles on the leaves and roots of this halophyte species PR:PROJECT_SUMMARY submitted to very high salinity stress, and the consequent use of single- and PR:PROJECT_SUMMARY multi-omics analysis strategies to study it. PR:INSTITUTE Embrapa Agroenergy PR:LAST_NAME Souza Júnior PR:FIRST_NAME Manoel Teixeira PR:ADDRESS Parque Estacao Biologica final Asa Norte Brasília DF 70770-901 BR, PQEB, sn - PR:ADDRESS Asa Norte, DF PR:EMAIL manoel.souza@embrapa.br PR:PHONE +55 (61) 3448-4246 #STUDY ST:STUDY_TITLE Osmoprotectants play a major role in the Portulaca oleracea resistance to high ST:STUDY_TITLE levels of salinity stress - Insights from a metabolomics and proteomics ST:STUDY_TITLE integrated approach ST:STUDY_SUMMARY Purslane is an invasive plant and is considered the eighth most common weed in ST:STUDY_SUMMARY the world. Because of that, its outdoor production in extensive areas faces ST:STUDY_SUMMARY several concerns. Kong & Zheng (2014) evaluated the potential of producing ST:STUDY_SUMMARY purslane in a hydroponic system, generating approximately 5.75 kg of fresh ST:STUDY_SUMMARY matter per m2 per month, which might yield 57.5 tons/hectare/year if cultivated ST:STUDY_SUMMARY in a bimestrial regime. The high productivity of purslane, when grown in ST:STUDY_SUMMARY controlled-environment agriculture, can open many opportunities for the purslane ST:STUDY_SUMMARY industry, even in the context of biosaline agriculture. Building up a robust ST:STUDY_SUMMARY multi-omics database on the response of purslane to salt stress and the ST:STUDY_SUMMARY subsequent study of it via an MOI analysis can create the basis for a future ST:STUDY_SUMMARY system biology approach to decode the genetics behind its resistance to salinity ST:STUDY_SUMMARY stress. The present study is a second step in building a robust database on the ST:STUDY_SUMMARY morpho-physiological and molecular responses of Portulaca oleracea L. to ST:STUDY_SUMMARY salinity stress and its subsequent use in attempting to decode the genetics ST:STUDY_SUMMARY behind its resistance to this abiotic stress. After reporting on the ST:STUDY_SUMMARY characterization of the morpho-physiological responses of young purslane plants ST:STUDY_SUMMARY to such stress using a robust salinization protocol, here we report a study on ST:STUDY_SUMMARY adult purslane plants through the characterization of the untargeted metabolome ST:STUDY_SUMMARY and proteome profiles on the leaves and roots of this halophyte species ST:STUDY_SUMMARY submitted to very high salinity stress, and the consequent use of single- and ST:STUDY_SUMMARY multi-omics analysis strategies to study it. ST:INSTITUTE Embrapa Agroenergy ST:LAST_NAME Souza Júnior ST:FIRST_NAME Manoel Teixeira ST:ADDRESS Parque Estacao Biologica final Asa Norte Brasília DF 70770-901 BR, PQEB, sn - ST:ADDRESS Asa Norte, DF ST:EMAIL manoel.souza@embrapa.br ST:PHONE +55 (61) 3448-4246 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Portulaca oleracea SU:TAXONOMY_ID 46147 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Purslane_CP1_Adult_Leaf_Polar_Positive Group:Leaf_Control RAW_FILE_NAME=Purslane_CP1_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP2_Adult_Leaf_Polar_Positive Group:Leaf_Control RAW_FILE_NAME=Purslane_CP2_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP3_Adult_Leaf_Polar_Positive Group:Leaf_Control RAW_FILE_NAME=Purslane_CP3_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP4_Adult_Leaf_Polar_Positive Group:Leaf_Control RAW_FILE_NAME=Purslane_CP4_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP1_Adult_Leaf_Polar_Negative Group:Leaf_Control RAW_FILE_NAME=Purslane_CP1_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP2_Adult_Leaf_Polar_Negative Group:Leaf_Control RAW_FILE_NAME=Purslane_CP2_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP3_Adult_Leaf_Polar_Negative Group:Leaf_Control RAW_FILE_NAME=Purslane_CP3_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP4_Adult_Leaf_Polar_Negative Group:Leaf_Control RAW_FILE_NAME=Purslane_CP4_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP1_Adult_Leaf_Lipidic_Positive Group:Leaf_Control RAW_FILE_NAME=Purslane_CP1_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP2_Adult_Leaf_Lipidic_Positive Group:Leaf_Control RAW_FILE_NAME=Purslane_CP2_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP3_Adult_Leaf_Lipidic_Positive Group:Leaf_Control RAW_FILE_NAME=Purslane_CP3_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP4_Adult_Leaf_Lipidic_Positive Group:Leaf_Control RAW_FILE_NAME=Purslane_CP4_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP1_Adult_Leaf_Lipidic_Negative Group:Leaf_Control RAW_FILE_NAME=Purslane_CP1_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP2_Adult_Leaf_Lipidic_Negative Group:Leaf_Control RAW_FILE_NAME=Purslane_CP2_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP3_Adult_Leaf_Lipidic_Negative Group:Leaf_Control RAW_FILE_NAME=Purslane_CP3_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP4_Adult_Leaf_Lipidic_Negative Group:Leaf_Control RAW_FILE_NAME=Purslane_CP4_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP1_Adult_Leaf_Polar_Positive Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP1_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP2_Adult_Leaf_Polar_Positive Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP2_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP3_Adult_Leaf_Polar_Positive Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP3_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP4_Adult_Leaf_Polar_Positive Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP4_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP1_Adult_Leaf_Polar_Negative Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP1_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP2_Adult_Leaf_Polar_Negative Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP2_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP3_Adult_Leaf_Polar_Negative Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP3_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP4_Adult_Leaf_Polar_Negative Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP4_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP1_Adult_Leaf_Lipidic_Positive Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP1_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP2_Adult_Leaf_Lipidic_Positive Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP2_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP3_Adult_Leaf_Lipidic_Positive Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP3_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP4_Adult_Leaf_Lipidic_Positive Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP4_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP1_Adult_Leaf_Lipidic_Negative Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP1_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP2_Adult_Leaf_Lipidic_Negative Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP2_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP3_Adult_Leaf_Lipidic_Negative Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP3_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP4_Adult_Leaf_Lipidic_Negative Group:Leaf_Stressed RAW_FILE_NAME=Purslane_SP4_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP1_Adult_Root_Lipidic_Negative Group:Root_Control RAW_FILE_NAME=Purslane_CP1_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP2_Adult_Root_Lipidic_Negative Group:Root_Control RAW_FILE_NAME=Purslane_CP2_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP3_Adult_Root_Lipidic_Negative Group:Root_Control RAW_FILE_NAME=Purslane_CP3_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP4_Adult_Root_Lipidic_Negative Group:Root_Control RAW_FILE_NAME=Purslane_CP4_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP1_Adult_Root_Lipidic_Positive Group:Root_Control RAW_FILE_NAME=Purslane_CP1_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP2_Adult_Root_Lipidic_Positive Group:Root_Control RAW_FILE_NAME=Purslane_CP2_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP3_Adult_Root_Lipidic_Positive Group:Root_Control RAW_FILE_NAME=Purslane_CP3_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP4_Adult_Root_Lipidic_Positive Group:Root_Control RAW_FILE_NAME=Purslane_CP4_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP1_Adult_Root_Polar_Negative Group:Root_Control RAW_FILE_NAME=Purslane_CP1_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP2_Adult_Root_Polar_Negative Group:Root_Control RAW_FILE_NAME=Purslane_CP2_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP3_Adult_Root_Polar_Negative Group:Root_Control RAW_FILE_NAME=Purslane_CP3_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP4_Adult_Root_Polar_Negative Group:Root_Control RAW_FILE_NAME=Purslane_CP4_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP1_Adult_Root_Polar_Positive Group:Root_Control RAW_FILE_NAME=Purslane_CP1_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP2_Adult_Root_Polar_Positive Group:Root_Control RAW_FILE_NAME=Purslane_CP2_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP3_Adult_Root_Polar_Positive Group:Root_Control RAW_FILE_NAME=Purslane_CP3_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_CP4_Adult_Root_Polar_Positive Group:Root_Control RAW_FILE_NAME=Purslane_CP4_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP1_Adult_Root_Lipidic_Negative Group:Root_Stressed RAW_FILE_NAME=Purslane_SP1_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP2_Adult_Root_Lipidic_Negative Group:Root_Stressed RAW_FILE_NAME=Purslane_SP2_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP3_Adult_Root_Lipidic_Negative Group:Root_Stressed RAW_FILE_NAME=Purslane_SP3_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP4_Adult_Root_Lipidic_Negative Group:Root_Stressed RAW_FILE_NAME=Purslane_SP4_Adult_Lipidic_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP1_Adult_Root_Lipidic_Positive Group:Root_Stressed RAW_FILE_NAME=Purslane_SP1_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP2_Adult_Root_Lipidic_Positive Group:Root_Stressed RAW_FILE_NAME=Purslane_SP2_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP3_Adult_Root_Lipidic_Positive Group:Root_Stressed RAW_FILE_NAME=Purslane_SP3_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP4_Adult_Root_Lipidic_Positive Group:Root_Stressed RAW_FILE_NAME=Purslane_SP4_Adult_Lipidic_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP1_Adult_Root_Polar_Negative Group:Root_Stressed RAW_FILE_NAME=Purslane_SP1_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP2_Adult_Root_Polar_Negative Group:Root_Stressed RAW_FILE_NAME=Purslane_SP2_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP3_Adult_Root_Polar_Negative Group:Root_Stressed RAW_FILE_NAME=Purslane_SP3_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP4_Adult_Root_Polar_Negative Group:Root_Stressed RAW_FILE_NAME=Purslane_SP4_Adult_Polar_Negative.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP1_Adult_Root_Polar_Positive Group:Root_Stressed RAW_FILE_NAME=Purslane_SP1_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP2_Adult_Root_Polar_Positive Group:Root_Stressed RAW_FILE_NAME=Purslane_SP2_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP3_Adult_Root_Polar_Positive Group:Root_Stressed RAW_FILE_NAME=Purslane_SP3_Adult_Polar_Positive.mzXML SUBJECT_SAMPLE_FACTORS - Purslane_SP4_Adult_Root_Polar_Positive Group:Root_Stressed RAW_FILE_NAME=Purslane_SP4_Adult_Polar_Positive.mzXML #COLLECTION CO:COLLECTION_SUMMARY Leaves and roots from both treatments - five replicates per treatment - were CO:COLLECTION_SUMMARY collected for biomass and mineral analysis. Fifteen samples of substrate were CO:COLLECTION_SUMMARY collected for mineral analysis, five before salinization, and ten at the end of CO:COLLECTION_SUMMARY the experiment - five from control and five from stressed plants. Leaves and CO:COLLECTION_SUMMARY roots from both treatments - five replicates per treatment - were collected and CO:COLLECTION_SUMMARY immediately immersed in liquid nitrogen and then stored at -80 °C until CO:COLLECTION_SUMMARY extraction of metabolites or proteins. CO:SAMPLE_TYPE Plant #TREATMENT TR:TREATMENT_SUMMARY The B1 accession of purslane (Portulaca oleracea L.) used in this study belongs TR:TREATMENT_SUMMARY to the Purslane Collection at Embrapa Agroenergia. Seeds underwent disinfection TR:TREATMENT_SUMMARY following the same procedure described in Belo Silva et al. (2022), which TR:TREATMENT_SUMMARY consisted of soaking in 2% sodium hypochlorite and Tween® 20 for five minutes, TR:TREATMENT_SUMMARY under slow agitation, and then washing with sterile water and drying on TR:TREATMENT_SUMMARY sterilized filter paper. After being seeded on a culture medium (MS 1/2 TR:TREATMENT_SUMMARY strength, Phytagel 0.2%, and pH 5.8) (Murashige and Skoog, 1962), it was kept TR:TREATMENT_SUMMARY for germination in a Growth chamber Conviron mod. Adaptis 1000TC (Controlled TR:TREATMENT_SUMMARY Environments Ltd, Winnipeg, Canada) at 150 μmol/m2/s of light and 30°C. After TR:TREATMENT_SUMMARY 13 days, seedlings were individually transferred to 200 ml plastic cups TR:TREATMENT_SUMMARY containing 100 g of sterilized substrate - clay soil, vermiculite, and a TR:TREATMENT_SUMMARY commercial substrate (Bioplant®), 2:1:1 (v:v:v) ratio – and transferred to TR:TREATMENT_SUMMARY another Conviron® growth chamber mod. PGW40 at 25±2°C, 500±20 μmol/m2/s of TR:TREATMENT_SUMMARY light, 65±5% air relative humidity, and photoperiod of 16/8 h (light/dark), and TR:TREATMENT_SUMMARY kept there until the end of the experiments. The plants were allowed to TR:TREATMENT_SUMMARY acclimatize for three days, and the salinity stress started three weeks after TR:TREATMENT_SUMMARY the end of the acclimatization period. The salinization experiment consisted of TR:TREATMENT_SUMMARY two salinity levels (0.0 and 2.0 g of NaCl / 100 g of the substrate), with 16 TR:TREATMENT_SUMMARY replicates (plants) in a completely randomized design, and the stress lasted 12 TR:TREATMENT_SUMMARY days. During the entire experiment, plants were at field capacity. To avoid the TR:TREATMENT_SUMMARY loss of Na+ or Cl-, no leakage of the saline solution was allowed to get out of TR:TREATMENT_SUMMARY the plastic cup, as described previously in Belo Silva et al. (2022). The water TR:TREATMENT_SUMMARY lost due to evapotranspiration was replaced with deionized water daily, and the TR:TREATMENT_SUMMARY electric conductivity at field capacity (ECfc) and water potential in the TR:TREATMENT_SUMMARY substrate solution was measured once - on the 8th day of stress - for all TR:TREATMENT_SUMMARY replicates. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For the metabolomics study, the following substances were acquired from Sigma SP:SAMPLEPREP_SUMMARY Aldrich (Merck, USA): methanol UHPLC grade, acetonitrile LC-MS grade, SP:SAMPLEPREP_SUMMARY methyl-tert-butyl-ether, formic acid LC-MS grade, and sodium hydroxide ACS SP:SAMPLEPREP_SUMMARY grade. Water was obtained using a Milli-Q system (Millipore, USA). Metabolites SP:SAMPLEPREP_SUMMARY were extracted using a well-established protocol, which provides polar and SP:SAMPLEPREP_SUMMARY lipidic fractions from the same samples. In this protocol, we first ground the SP:SAMPLEPREP_SUMMARY plant material (roots or leaves) in a ball mill (Biospec Products, USA), then SP:SAMPLEPREP_SUMMARY added to a microtube containing 1 mL from a solution (1:3) of methanol and SP:SAMPLEPREP_SUMMARY methyl-tert-butyl-ether at -20°C. Samples were incubated for 10 min at 4.0 °C, SP:SAMPLEPREP_SUMMARY then ultrasonicated for another 10 min in an ice bath. A solution (1:3) of SP:SAMPLEPREP_SUMMARY methanol and water was added to each microtube and then submitted to SP:SAMPLEPREP_SUMMARY centrifugation (12,000 rpm at 4.0°C for 5 min). The polar (upper) and non-polar SP:SAMPLEPREP_SUMMARY (lower) fractions were collected and vacuum-dried in a speed vac system SP:SAMPLEPREP_SUMMARY overnight (Centrivap, Labconco, Kansas City, MO, USA). Four microliters of the SP:SAMPLEPREP_SUMMARY extract were resuspended in 850 μL of the methanol and water (1:3) solvent SP:SAMPLEPREP_SUMMARY mixture and then analyzed by UHPLC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Waters ACQUITY UPLC BEH C8 (150 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT isocratic at the start (0 - 0.5 min) with 4% of B solvent, then at linear CH:FLOW_GRADIENT gradient (0.5 – 10 min) with 34% B and (10 – 15 min) with 100% B, and CH:FLOW_GRADIENT finally isocratic (15 – 18 min) with 100% B CH:FLOW_RATE 400 μL min−1 CH:COLUMN_TEMPERATURE 40 °C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker maXis Impact qTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS For external calibration, we used a sodium formate solution (10 mM NaOH solution MS:MS_COMMENTS in 50/50 v/v isopropanol/water containing 0.2% formic acid) directly injected MS:MS_COMMENTS through a 6-port valve at the beginning of each chromatographic run. UHPLC-MS MS:MS_COMMENTS data was acquired by the HyStar Application version 3.2 (Bruker Daltonics, MS:MS_COMMENTS Germany). Data pre-processing was performed using the software DataAnalysis MS:MS_COMMENTS version 4.4 (Bruker Daltonics, Germany), where raw data from the UHPLC-MS MS:MS_COMMENTS analysis were exported as .mzXML files. MS:MS_RESULTS_FILE ST002537_AN004176_Results.txt UNITS:m/z Has m/z:Yes Has RT:No RT units:No RT data MS:MS_RESULTS_FILE ST002537_AN004176_Results.txt UNITS:m/z Has m/z:Yes Has RT:No RT units:No RT data MS:MS_RESULTS_FILE ST002537_AN004176_Results.txt UNITS:m/z Has m/z:Yes Has RT:No RT units:No RT data MS:MS_RESULTS_FILE ST002537_AN004176_Results.txt UNITS:m/z Has m/z:Yes Has RT:No RT units:No RT data #END