#METABOLOMICS WORKBENCH Cristina_20200425_024934 DATATRACK_ID:1993 STUDY_ID:ST001371 ANALYSIS_ID:AN002289 PROJECT_ID:PR000937 VERSION 1 CREATED_ON May 5, 2020, 12:52 pm #PROJECT PR:PROJECT_TITLE Untargeted metabolomics of Quercus ilex acorns PR:PROJECT_TYPE LC-MSE analysis PR:PROJECT_SUMMARY UPLC-MSE analysis of samples from Quercus ilex acorns flour. The objective of PR:PROJECT_SUMMARY the study is to obtain a metabolomic profile of several acorns from different PR:PROJECT_SUMMARY trees. This phytochemical analysis and characterization will be a base for PR:PROJECT_SUMMARY identification of bioactive, antinutritional, or toxic compounds and PR:PROJECT_SUMMARY traceability analysis. PR:INSTITUTE Universidad de Córdoba PR:DEPARTMENT Department Biochemistry and Molecular Biology PR:LABORATORY Agroforestry and Plant Biochemistry, Proteomics and Systems Biology PR:LAST_NAME López-Hidalgo PR:FIRST_NAME Cristina PR:ADDRESS Campus de Rabanales; Edificio C6, Planta Baja PR:EMAIL n12lohic@uco.es PR:PHONE 626894948 PR:FUNDING_SOURCE This work was supported by the University of Cordoba and financial support from PR:FUNDING_SOURCE the Spanish Ministry of Economy and Competitiveness (Project BIO2015-64737-R2). PR:PUBLICATIONS Phytochemical composition and variability among Quercus ilex acorns determined PR:PUBLICATIONS by NIRS and MS-based approaches #STUDY ST:STUDY_TITLE Untargeted metabolomics of Quercus ilex acorns ST:STUDY_TYPE LC-MSE analysis ST:STUDY_SUMMARY UPLC-MSE analysis of samples from Quercus ilex acorns flour. The objective of ST:STUDY_SUMMARY the study is to obtain a metabolomic profile of several acorns from different ST:STUDY_SUMMARY trees. This phytochemical analysis and characterization will be a base for the ST:STUDY_SUMMARY identification of bioactive, antinutritional, or toxic compounds and ST:STUDY_SUMMARY traceability analysis. ST:INSTITUTE Universidad de Córdoba ST:DEPARTMENT Department Biochemistry and Molecular Biology ST:LABORATORY Agroforestry and Plant Biochemistry, Proteomics and Systems Biology ST:LAST_NAME López-Hidalgo ST:FIRST_NAME Cristina ST:ADDRESS Campus de Rabanales; Edificio C6, Planta Baja ST:EMAIL n12lohic@uco.es ST:PHONE 626894948 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Quercus ilex SU:TAXONOMY_ID 58334 #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 - B F_POS C Mixture:Blank RAW_FILE_NAME=B F_POS C.mzXML SUBJECT_SAMPLE_FACTORS - B_POS C1 Mixture:Blank RAW_FILE_NAME=B_POS C1.mzXML SUBJECT_SAMPLE_FACTORS - B_POS C2 Mixture:Blank RAW_FILE_NAME=B_POS C2.mzXML SUBJECT_SAMPLE_FACTORS - B_POS C3 Mixture:Blank RAW_FILE_NAME=B_POS C3.mzXML SUBJECT_SAMPLE_FACTORS - B_POS C4 Mixture:Blank RAW_FILE_NAME=B_POS C4.mzXML SUBJECT_SAMPLE_FACTORS - B_POS C5 Mixture:Blank RAW_FILE_NAME=B_POS C5.mzXML SUBJECT_SAMPLE_FACTORS - BOL1_POS C Mixture:BOL RAW_FILE_NAME=BOL1_POS C.mzXML SUBJECT_SAMPLE_FACTORS - BOL2_POS C Mixture:BOL RAW_FILE_NAME=BOL2_POS C.mzXML SUBJECT_SAMPLE_FACTORS - BOL3_POS C Mixture:BOL RAW_FILE_NAME=BOL3_POS C.mzXML SUBJECT_SAMPLE_FACTORS - BR_POS C Mixture:Blank RAW_FILE_NAME=BR_POS C.mzXML SUBJECT_SAMPLE_FACTORS - BR_POS C_1 Mixture:Blank RAW_FILE_NAME=BR_POS C_1.mzXML SUBJECT_SAMPLE_FACTORS - MAD1_POS C Mixture:MAD RAW_FILE_NAME=MAD1_POS C.mzXML SUBJECT_SAMPLE_FACTORS - MAD2_POS C Mixture:MAD RAW_FILE_NAME=MAD2_POS C.mzXML SUBJECT_SAMPLE_FACTORS - MAD3_POS C Mixture:MAD RAW_FILE_NAME=MAD3_POS C.mzXML SUBJECT_SAMPLE_FACTORS - MIX FEN F_POS C Mixture:Phenolics mixture RAW_FILE_NAME=MIX FEN F_POS C.mzXML SUBJECT_SAMPLE_FACTORS - MIX FEN_POS C Mixture:Phenolics mixture RAW_FILE_NAME=MIX FEN_POS C.mzXML SUBJECT_SAMPLE_FACTORS - NTC1_POS C Mixture:NTC RAW_FILE_NAME=NTC1_POS C.mzXML SUBJECT_SAMPLE_FACTORS - NTC2_POS C Mixture:NTC RAW_FILE_NAME=NTC2_POS C.mzXML SUBJECT_SAMPLE_FACTORS - NTC3_POS C Mixture:NTC RAW_FILE_NAME=NTC3_POS C.mzXML SUBJECT_SAMPLE_FACTORS - POL1_POS C Mixture:POL RAW_FILE_NAME=POL1_POS C.mzXML SUBJECT_SAMPLE_FACTORS - POL2_POS C Mixture:POL RAW_FILE_NAME=POL2_POS C.mzXML SUBJECT_SAMPLE_FACTORS - POL3_POS C Mixture:POL RAW_FILE_NAME=POL3_POS C.mzXML SUBJECT_SAMPLE_FACTORS - QC MIX2_POS C01 Mixture:Quality Control mixture RAW_FILE_NAME=QC MIX2_POS C01.mzXML SUBJECT_SAMPLE_FACTORS - QC MIX2_POS C02 Mixture:Quality Control mixture RAW_FILE_NAME=QC MIX2_POS C02.mzXML SUBJECT_SAMPLE_FACTORS - QC MIX2_POS C03 Mixture:Quality Control mixture RAW_FILE_NAME=QC MIX2_POS C03.mzXML SUBJECT_SAMPLE_FACTORS - QC MIX2_POS C04 Mixture:Quality Control mixture RAW_FILE_NAME=QC MIX2_POS C04.mzXML SUBJECT_SAMPLE_FACTORS - B F_NEG C Mixture:Blank RAW_FILE_NAME=B F_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - B_NEG C1 Mixture:Blank RAW_FILE_NAME=B_NEG C1.mzXML SUBJECT_SAMPLE_FACTORS - B_NEG C2 Mixture:Blank RAW_FILE_NAME=B_NEG C2.mzXML SUBJECT_SAMPLE_FACTORS - B_NEG C3 Mixture:Blank RAW_FILE_NAME=B_NEG C3.mzXML SUBJECT_SAMPLE_FACTORS - B_NEG C4 Mixture:Blank RAW_FILE_NAME=B_NEG C4.mzXML SUBJECT_SAMPLE_FACTORS - B_NEG C5 Mixture:Blank RAW_FILE_NAME=B_NEG C5.mzXML SUBJECT_SAMPLE_FACTORS - BOL1_NEG C Mixture:BOL RAW_FILE_NAME=BOL1_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - BOL2_NEG C Mixture:BOL RAW_FILE_NAME=BOL2_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - BOL3_NEG C Mixture:BOL RAW_FILE_NAME=BOL3_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - BR_NEG C Mixture:Blank RAW_FILE_NAME=BR_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - BR_NEG C_1 Mixture:Blank RAW_FILE_NAME=BR_NEG C_1.mzXML SUBJECT_SAMPLE_FACTORS - MAD1_NEG C Mixture:MAD RAW_FILE_NAME=MAD1_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - MAD2_NEG C Mixture:MAD RAW_FILE_NAME=MAD2_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - MAD3_NEG C Mixture:MAD RAW_FILE_NAME=MAD3_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - MIX FEN F_NEG C Mixture:Phenolics mixture RAW_FILE_NAME=MIX FEN F_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - MIX FEN_NEG C Mixture:Phenolics mixture RAW_FILE_NAME=MIX FEN_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - NTC1_NEG C Mixture:NTC RAW_FILE_NAME=NTC1_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - NTC2_NEG C Mixture:NTC RAW_FILE_NAME=NTC2_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - NTC3_NEG C Mixture:NTC RAW_FILE_NAME=NTC3_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - POL1_NEG C Mixture:POL RAW_FILE_NAME=POL1_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - POL2_NEG C Mixture:POL RAW_FILE_NAME=POL2_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - POL3_NEG C Mixture:POL RAW_FILE_NAME=POL3_NEG C.mzXML SUBJECT_SAMPLE_FACTORS - QC MIX2_NEG C01 Mixture:Quality Control mixture RAW_FILE_NAME=QC MIX2_NEG C01.mzXML SUBJECT_SAMPLE_FACTORS - QC MIX2_NEG C02 Mixture:Quality Control mixture RAW_FILE_NAME=QC MIX2_NEG C02.mzXML SUBJECT_SAMPLE_FACTORS - QC MIX2_NEG C03 Mixture:Quality Control mixture RAW_FILE_NAME=QC MIX2_NEG C03.mzXML SUBJECT_SAMPLE_FACTORS - QC MIX2_NEG C04 Mixture:Quality Control mixture RAW_FILE_NAME=QC MIX2_NEG C04.mzXML #COLLECTION CO:COLLECTION_SUMMARY Sample collection: Mature acorns from holm oak (Quercus ilex L. subsp.ballota CO:COLLECTION_SUMMARY [Desf.] Samp.) were collected from four different trees located in Aldea de CO:COLLECTION_SUMMARY Cuenca (province of Cordoba, Andalusia, Spain). All acorns were picked at the CO:COLLECTION_SUMMARY optimal harvest maturity window on the same day. Sorting, disinfection, and CO:COLLECTION_SUMMARY storage of healthy acorns were conducted according to Bonner & Vozzo, (1987). CO:COLLECTION_SUMMARY Acorn flour preparation: Healthy acorns (20 units per tree) were scarified with CO:COLLECTION_SUMMARY a knife by making transversal and longitudinal cuts, thus permitting the CO:COLLECTION_SUMMARY pericarp to be rapidly removed. Flour was prepared by seed (without seed coat) CO:COLLECTION_SUMMARY grinding with liquid nitrogen in a blade mill (IKA Dry Mill Basic A10) until a CO:COLLECTION_SUMMARY powder was obtained (Valero Galván, Jorrín Novo, Cabrera, et al., 2012). Flour CO:COLLECTION_SUMMARY was lyophilized and then macerated in a mortar until a fine powder was obtained. CO:COLLECTION_SUMMARY Samples were stored at 4 ºC in within a desiccator, in darkness, until NIRS CO:COLLECTION_SUMMARY analysis or metabolite extraction. CO:SAMPLE_TYPE Acorns CO:STORAGE_CONDITIONS Room temperature #TREATMENT TR:TREATMENT_SUMMARY Mature acorns from holm oak (Quercus ilex L. subsp.ballota [Desf.] Samp.) were TR:TREATMENT_SUMMARY collected from nine different trees. From these, four were selected for TR:TREATMENT_SUMMARY metabolomic analysis (POL, BOL, NTC, and MAD). TR:PLANT_GROWTH_LOCATION Aldea de Cuenca (province of Cordoba, Andalusia, Spain) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from acorn flour as described by Valledor et al., SP:SAMPLEPREP_SUMMARY (2014), with minor modifications. 600 µL of ice-cold methanol: chloroform: SP:SAMPLEPREP_SUMMARY water (5:2:2) was added to 50 mg of acorn flour, mixed by vortexing, and the SP:SAMPLEPREP_SUMMARY mixture sonicated (ultrasonic bath, 40 kHZ for 10 min). After centrifugation (4 SP:SAMPLEPREP_SUMMARY oC, 4 min, 20,000 × g), the pellet was once more extracted with 200 µL of cold SP:SAMPLEPREP_SUMMARY methanol: chloroform: water (5:2:2). The two supernatants were combined and SP:SAMPLEPREP_SUMMARY vacuum dried at 30 oC (Speedvac, Eppendorf Vacuum Concentrator Plus/5301). Dried SP:SAMPLEPREP_SUMMARY extracts were reconstituted in methanol, centrifuged at 20,000 × g for 10 min, SP:SAMPLEPREP_SUMMARY filtered through 0.22 µm PTPE membranes (Thermo Scientific, MA, USA) and SP:SAMPLEPREP_SUMMARY filtrate collected in 1.5 mL LC/MS certified sample vials. SP:PROCESSING_STORAGE_CONDITIONS Room temperature SP:EXTRACT_STORAGE On ice SP:SAMPLE_RESUSPENSION Methanol #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY MS analysis was conducted in an ultra-performance liquid chromatography (UPLC CH:CHROMATOGRAPHY_SUMMARY Acquity H-Class, Waters, Milford, USA) coupled to a quadrupole time of flight CH:CHROMATOGRAPHY_SUMMARY (QTof) G2-XS mass spectrometer (Waters, Milford, USA). The chromatographic CH:CHROMATOGRAPHY_SUMMARY separation was carried out in a C18 column (2.1×100 mm, 1.7 µm, Waters, CH:CHROMATOGRAPHY_SUMMARY Milford, USA), kept at 45 °C. The injection volume was 5 µL, and the flow rate CH:CHROMATOGRAPHY_SUMMARY set at 0.450 mL min−1. Mobile phases consisted of 0.1 % formic acid in Milli-Q CH:CHROMATOGRAPHY_SUMMARY water (A) and methanol (B). The gradient elution profile was as follow (time, % CH:CHROMATOGRAPHY_SUMMARY B): 0 min, 2% B; 0.25 min, 2 % B; 12.25 min, 99 % B; 13.0 min, 99 % B; 13.01 CH:CHROMATOGRAPHY_SUMMARY min; 2 % B; 17.00 min; 2 % B and then the column was equilibrated for 5 min CH:CHROMATOGRAPHY_SUMMARY prior to each analysis. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity H-Class CH:COLUMN_NAME C18 column (2.1×100 mm, 1.7 µm, Waters, Milford, USA) CH:FLOW_RATE 0.450 mL min−1 CH:COLUMN_TEMPERATURE 45 CH:SOLVENT_A 0.1 % formic acid in Milli-Q water CH:SOLVENT_B 0.1 % formic acid in methanol CH:SAMPLE_INJECTION 5 CH:ANALYTICAL_TIME 17 min CH:TIME_PROGRAM 17 min #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Agroforestry and Plant Biochemistry, Proteomics and Systems Biology AN:DETECTOR_TYPE QTOF AN:DATA_FORMAT .raw and .mzXML #MS MS:INSTRUMENT_NAME Waters Synapt G2 XS QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS MS analysis was conducted in an ultra-performance liquid chromatography (UPLC MS:MS_COMMENTS Acquity H-Class, Waters, Milford, USA) coupled to a quadrupole time of flight MS:MS_COMMENTS (QTof) G2-XS mass spectrometer (Waters, Milford, USA). The chromatographic MS:MS_COMMENTS separation was carried out in a C18 column (2.1×100 mm, 1.7 µm, Waters, MS:MS_COMMENTS Milford, USA), kept at 45 °C. The injection volume was 5 µL, and the flow rate MS:MS_COMMENTS set at 0.450 mL min−1. Mobile phases consisted of 0.1 % formic acid in Milli-Q MS:MS_COMMENTS water (A) and methanol (B). The gradient elution profile was as follow (time, % MS:MS_COMMENTS B): 0 min, 2% B; 0.25 min, 2 % B; 12.25 min, 99 % B; 13.0 min, 99 % B; 13.01 MS:MS_COMMENTS min; 2 % B; 17.00 min; 2 % B and then the column was equilibrated for 5 min MS:MS_COMMENTS prior to each analysis. The MS acquisition was performed in negative and MS:MS_COMMENTS positive ionization modes in a scan range from m/z 100 to 1200 and time MS:MS_COMMENTS acquisition of 0 to 17 min. The analysis type performed was accurate mass MS:MS_COMMENTS screening on MSE data with a low collision energy of 4.00 eV and a high-energy MS:MS_COMMENTS ramp of 10.00 to 45.00 eV. The capillary and cone voltage were set at 2.50 kV MS:MS_COMMENTS and 40 V, respectively. The desolvation gas was set to 600 L h−1, the cone gas MS:MS_COMMENTS set to 50 L h−1 and the source and desolvation temperature was set to 100 °C MS:MS_COMMENTS and 250 °C, respectively. For automated accurate mass measurement, a solution MS:MS_COMMENTS of leucine-enkephalin (200 ng mL−1) in methanol: water (50:50) with 0.1% MS:MS_COMMENTS formic acid was used as lock mass and pumped at a flow rate of 5 µL min−1. MS:MS_COMMENTS The molecule of leucine-enkephalin (m/z 556.2766 in ESI+ and m/z 554.262 in MS:MS_COMMENTS ESI−) was used for recalibrating the mass axis and ensuring a robust accurate MS:MS_COMMENTS mass measurement at any time. For continuous quality assurance and to provide MS:MS_COMMENTS confidence in the data, quality control (QC), a mix prepared from equal volumes MS:MS_COMMENTS of all the samples was injected between every three samples in the batch along MS:MS_COMMENTS with methanol as a blank run to correct a drift of the raw signal intensity MS:MS_COMMENTS during the analysis. All the data acquired were exported by Waters UNIFI MS:MS_COMMENTS software in order to analyze by the software Progenesis QI (Nonlinear Dynamics, MS:MS_COMMENTS Newcastle, United Kingdom). MS:COLLISION_ENERGY 4.00 eV MS:FRAGMENT_VOLTAGE High-ernergy ramp of 10.00 to 45.00 eV MS:FRAGMENTATION_METHOD MSE MS:MS_RESULTS_FILE ST001371_AN002289_Results.txt UNITS:abundance Has m/z:Yes Has RT:Yes RT units:Minutes #END