{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001934","ANALYSIS_ID":"AN003144","VERSION":"1","CREATED_ON":"September 24, 2021, 1:56 pm"},

"PROJECT":{"PROJECT_TITLE":"Teasing out the Species- and Organ-Specific Patterns of Flavonoid Glycosides, Styrylpyrones, Caffeic Acid Esters, and Coumarins in Equisetum","PROJECT_SUMMARY":"Members of the genus Equisetum are often referred to as “living fossils”, partly because they are the only extant representatives of the Equisetidae, a subclass that was once prominent in late Paleozoic forests. Several classes of specialized metabolites have been reported to occur in the genus Equisetum. However, while steady progress is being made with identifying individual novel metabolites of Equisetum, few if any analyses have focused on assessing the chemical diversity across the genus. The present study focused on three species: E. hyemale subsp. affine (rough horsetail or scouring rush), which is native to the temperate to artic portions of North America; E. arvense (common horsetail), which is endemic to the arctic and temperate regions of the northern hemisphere; and Equisetum telmateia subsp. braunii (Milde) Hauke (giant horsetail), which is native to western North America. Both below-ground rhizome and above-ground shoot material was harvested from each species, extracted with aqueous methanol, and subjected to non-targeted HPLC-QTOF-MS analysis. This research project was designed to lay the foundation for continued research to capture the metabolic capabilities in the ferns and fern allies.","INSTITUTE":"Washington State University","DEPARTMENT":"Institute of Biological Chemistry","LABORATORY":"Lange","LAST_NAME":"Lange","FIRST_NAME":"Mark","ADDRESS":"Plant Sciences Building, Pullman, Washington 99164","EMAIL":"lange-m@wsu.edu","PHONE":"+1-509-335-3794"},

"STUDY":{"STUDY_TITLE":"Teasing out the Species- and Organ-Specific Patterns of Flavonoid Glycosides, Styrylpyrones, Caffeic Acid Esters, and Coumarins in Equisetum","STUDY_SUMMARY":"Members of the genus Equisetum are often referred to as “living fossils”, partly because they are the only extant representatives of the Equisetidae, a subclass that was once prominent in late Paleozoic forests. Several classes of specialized metabolites have been reported to occur in the genus Equisetum. However, while steady progress is being made with identifying individual novel metabolites of Equisetum, few if any analyses have focused on assessing the chemical diversity across the genus. The present study focused on three species: E. hyemale subsp. affine (rough horsetail or scouring rush), which is native to the temperate to artic portions of North America; E. arvense (common horsetail), which is endemic to the arctic and temperate regions of the northern hemisphere; and Equisetum telmateia subsp. braunii (Milde) Hauke (giant horsetail), which is native to western North America. Both below-ground rhizome and above-ground shoot material was harvested from each species, extracted with aqueous methanol, and subjected to non-targeted HPLC-QTOF-MS analysis. This research project was designed to lay the foundation for continued research to capture the metabolic capabilities in the ferns and fern allies.","INSTITUTE":"Washington State University","DEPARTMENT":"Institute of Biological Chemistry","LABORATORY":"Lange","LAST_NAME":"Lange","FIRST_NAME":"Mark","ADDRESS":"Plant Sciences Building, Pullman, Washington 99164","EMAIL":"lange-m@wsu.edu","PHONE":"+1-509-335-3794","NUM_GROUPS":"6","TOTAL_SUBJECTS":"30"},

"SUBJECT":{"SUBJECT_TYPE":"Plant","SUBJECT_SPECIES":"Equisetum arvense;Equisetum hyemale;Equisetum telmateia"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Ea_rhiz_1",
"Factors":{"Species":"E. arvense","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"EA.R1.d"}
},
{
"Subject ID":"-",
"Sample ID":"Ea_rhiz_2",
"Factors":{"Species":"E. arvense","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"EA.R2.d"}
},
{
"Subject ID":"-",
"Sample ID":"Ea_rhiz_3",
"Factors":{"Species":"E. arvense","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"EA.R3.d"}
},
{
"Subject ID":"-",
"Sample ID":"Ea_rhiz_4",
"Factors":{"Species":"E. arvense","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"EA.R4.d"}
},
{
"Subject ID":"-",
"Sample ID":"Ea_rhiz_5",
"Factors":{"Species":"E. arvense","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"EA.R5.d"}
},
{
"Subject ID":"-",
"Sample ID":"Ea_stem_1",
"Factors":{"Species":"E. arvense","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"EA.S1.d"}
},
{
"Subject ID":"-",
"Sample ID":"Ea_stem_2",
"Factors":{"Species":"E. arvense","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"EA.S2.d"}
},
{
"Subject ID":"-",
"Sample ID":"Ea_stem_3",
"Factors":{"Species":"E. arvense","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"EA.S3.d"}
},
{
"Subject ID":"-",
"Sample ID":"Ea_stem_4",
"Factors":{"Species":"E. arvense","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"EA.S4.d"}
},
{
"Subject ID":"-",
"Sample ID":"Ea_stem_5",
"Factors":{"Species":"E. arvense","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"EA.S5.d"}
},
{
"Subject ID":"-",
"Sample ID":"Eh_rhiz_1",
"Factors":{"Species":"E. hyemale","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"EH.R1.d"}
},
{
"Subject ID":"-",
"Sample ID":"Eh_rhiz_2",
"Factors":{"Species":"E. hyemale","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"EH.R2.d"}
},
{
"Subject ID":"-",
"Sample ID":"Eh_rhiz_3",
"Factors":{"Species":"E. hyemale","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"EH.R3.d"}
},
{
"Subject ID":"-",
"Sample ID":"Eh_rhiz_4",
"Factors":{"Species":"E. hyemale","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"EH.R4.d"}
},
{
"Subject ID":"-",
"Sample ID":"Eh_rhiz_5",
"Factors":{"Species":"E. hyemale","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"EH.R5.d"}
},
{
"Subject ID":"-",
"Sample ID":"Eh_stem_1",
"Factors":{"Species":"E. hyemale","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"EH.S1.d"}
},
{
"Subject ID":"-",
"Sample ID":"Eh_stem_2",
"Factors":{"Species":"E. hyemale","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"EH.S2.d"}
},
{
"Subject ID":"-",
"Sample ID":"Eh_stem_3",
"Factors":{"Species":"E. hyemale","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"EH.S3.d"}
},
{
"Subject ID":"-",
"Sample ID":"Eh_stem_4",
"Factors":{"Species":"E. hyemale","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"EH.S4.d"}
},
{
"Subject ID":"-",
"Sample ID":"Eh_stem_5",
"Factors":{"Species":"E. hyemale","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"EH.S5.d"}
},
{
"Subject ID":"-",
"Sample ID":"Et_rhiz_1",
"Factors":{"Species":"E. telmateia","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"ET.R1.d"}
},
{
"Subject ID":"-",
"Sample ID":"Et_rhiz_2",
"Factors":{"Species":"E. telmateia","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"ET.R2.d"}
},
{
"Subject ID":"-",
"Sample ID":"Et_rhiz_3",
"Factors":{"Species":"E. telmateia","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"ET.R3.d"}
},
{
"Subject ID":"-",
"Sample ID":"Et_rhiz_4",
"Factors":{"Species":"E. telmateia","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"ET.R4.d"}
},
{
"Subject ID":"-",
"Sample ID":"Et_rhiz_5",
"Factors":{"Species":"E. telmateia","Organ":"rhizome"},
"Additional sample data":{"RAW_FILE_NAME":"ET.R5.d"}
},
{
"Subject ID":"-",
"Sample ID":"Et_stem_1",
"Factors":{"Species":"E. telmateia","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"ET.S1.d"}
},
{
"Subject ID":"-",
"Sample ID":"Et_stem_2",
"Factors":{"Species":"E. telmateia","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"ET.S2.d"}
},
{
"Subject ID":"-",
"Sample ID":"Et_stem_3",
"Factors":{"Species":"E. telmateia","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"ET.S3.d"}
},
{
"Subject ID":"-",
"Sample ID":"Et_stem_4",
"Factors":{"Species":"E. telmateia","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"ET.S4.d"}
},
{
"Subject ID":"-",
"Sample ID":"Et_stem_5",
"Factors":{"Species":"E. telmateia","Organ":"stem"},
"Additional sample data":{"RAW_FILE_NAME":"ET.S5.d"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"E. arvense, E. hyemale and E. telmateia (voucher specimens deposited with the  John G. Searle Herbarium of the Field Museum, Chicago, IL, USA) were maintained  in a greenhouse under ambient lighting, with supplemental lighting from sodium- vapor lamps. The photosynthetically active radiation varied from 15 to 25 mol  m-2 d-1. Temperatures ranged between 22 and 27 °C and the humidity was set to 70  ± 10 %. Five biological replicates (separate plants) were harvested at the same  time of day for below-ground rhizomes and above-ground stems of vegetative  shoots. Samples were snap-frozen in liquid nitrogen and freeze-dried (aerial  parts for 5 days, rhizomes for 7 days). Lyophilized material was submerged in  liquid nitrogen, homogenized using mortar and pestle.","SAMPLE_TYPE":"Tissue homogenate","STORAGE_CONDITIONS":"-80 °C"},

"TREATMENT":{"TREATMENT_SUMMARY":"No Treatment"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Five biological replicates (separate plants) were harvested at the same  time of day for below-ground rhizomes and above-ground stems of vegetative  shoots. Samples were snap-frozen in liquid nitrogen and freeze-dried (aerial  parts for 5 days, rhizomes for 7 days). Lyophilized material was submerged in  liquid nitrogen, homogenized using mortar and pestle.","EXTRACTION_METHOD":"Frozen tissue homogenate (30 mg per sample) was transferred to a 2 ml reaction  tube and extracted with 1 ml of 80 % aqueous methanol (containing 10 mg/l  anthracene-9-carboxylic acid as internal standard) by vigorous shaking (VX-2500  multi-tube vortexer, VWR Scientific, South Plainfield, NY, USA) for 10 min and  subsequent sonication for 20 min (FS30 ultrasonic cleaner, Fisher Scientific,  Hampton, NY, USA). Following centrifugation for 10 min at 13,000 × g (5415  microfuge, Eppendorf, Enfield, CT, USA), the supernatant was filtered through  0.22 μm polypropylene syringe filter tips, and the flow-through collected in  plastic inserts for 2 ml reaction vials."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HPLC","INSTRUMENT_NAME":"Agilent 1290 HPLC","COLUMN_NAME":"HD Zorbax SB-Aq (100 × 2.1 × mm, 1.8 µm)","FLOW_GRADIENT":"5 % B to 10 % B at 5 min, 20 % B at 10 min, 80 % B at 35 min, 95 % B at 45 min","FLOW_RATE":"0.6 ml/min","COLUMN_TEMPERATURE":"60 °C","SOLVENT_A":"0.1 % formic acid in water","SOLVENT_B":"0.1 % formic acid in acetonitrile","INTERNAL_STANDARD":"10 mg/l Anthracene-9-carboxylic acid","SAMPLE_INJECTION":"10 ul","TARGET_SAMPLE_TEMPERATURE":"Autosampler set to 4 °C"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Lange","OPERATOR_NAME":"Parrish","SOFTWARE_VERSION":"MassHunter Qualitative Analysis software version B.07.00 Service Pack 1 build 7.0.7024.29, Profinder B.06.00 build 6.0.0625.0"},

"MS":{"INSTRUMENT_NAME":"Agilent 6530 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"HPLC–QTOF–MS with electrospray ion source (positive ion mode) Raw data sets were opened in the Profinder B.06.00 build 6.0.0625.0 software package (Agilent Technologies, Santa Clara, CA, USA) and molecular feature elements (MFEs) obtained using the Batch Recursive Feature Extraction algorithm. Binning and alignment tolerances were set to 10 % + 20 s for the retention time and 10 ppm + 2 mDa for the mass accuracy, and 0.0025 m/z + 5.0 ppm for the isotope grouping space tolerance. Additional parameters that were considered for feature extraction were quasi-molecular ions and adducts ([M+H]+, [M+Na]+, [M+K]+, [M+NH4]+), dimers, neutral losses (H2O, H3PO4, C6H10O5 (glucose), C12H20O9 (rutinose), C12H20O10 (sophorose), C6H10O4 (rhamnose), and C5H8O4 (xylose)), absolute peak height ≥ 2000 counts, and occurrence required in a minimum of four of the five replicates of each sample type. These pre-processing steps generated 848 MFEs (849 including ISTD), and exported into an Excel spreadsheet. Additional exclusion criteria for MFEs were: relative standard deviation of mass accuracy  greater than 5.0 ppm; percent relative standard deviation returned as ”NaN” (Not a Number) or an empty cell; an unacceptably close accurate mass and retention time (± 0.010 m/z and ± 0.02 min.; screened as duplicates); or if it was a fragment. This additional filtering returned 544 remaining MFEs. Peak areas of MFEs for each sample were normalized based on sample weight and the peak area of the internal standard (MFEs without a peak area were filled in with a nominal value of two).","SOURCE_TEMPERATURE":"325 °C","DATAFORMAT":".d","NEBULIZER":"2.4 bar","MS_RESULTS_FILE":"ST001934_AN003144_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes"}

}