#METABOLOMICS WORKBENCH tsimaom_20230417_055303 DATATRACK_ID:3866 STUDY_ID:ST002570 ANALYSIS_ID:AN004234 PROJECT_ID:PR001657 VERSION 1 CREATED_ON April 20, 2023, 10:02 am #PROJECT PR:PROJECT_TITLE Plant lipidomics studies PR:PROJECT_TYPE MS untargeted analysis PR:PROJECT_SUMMARY Untargeted analysis on Bromeliades leaf samples. The aim of this study was to PR:PROJECT_SUMMARY preliminary evaluate the lipid profile alterations on Pitcairnia flammea leaves PR:PROJECT_SUMMARY based on variations in solvent proportion and ultrasonic ice bath extraction PR:PROJECT_SUMMARY time, followed by a lipid class-enriched analysis employing chemometric PR:PROJECT_SUMMARY techniques. PR:INSTITUTE University of Campinas PR:DEPARTMENT Chemistry's Institute PR:LABORATORY Laboratory of Bioanalytics and Integrated Omics PR:LAST_NAME Matos PR:FIRST_NAME Taynara PR:ADDRESS Rua Josué de Castro, s/n – Cidade Universitária, 13083-970, Campinas – SP, PR:ADDRESS Brazil PR:EMAIL t262827@dac.unicamp.br PR:PHONE (85)996154192 #STUDY ST:STUDY_TITLE Evaluation of Extraction Parameters for the Analysis of Lipid Classes in Plants ST:STUDY_SUMMARY The aim of this study was to preliminary evaluate the lipid profile alterations ST:STUDY_SUMMARY on Pitcairnia flammea leaves based on variations in solvent proportion and ST:STUDY_SUMMARY ultrasonic ice bath extraction time, followed by a lipid class-enriched analysis ST:STUDY_SUMMARY employing chemometric techniques. In the plant extraction method employed, the ST:STUDY_SUMMARY sample was separated in 3 phases 2 : organic, aqueous and protein phases. The ST:STUDY_SUMMARY parameters evaluated in our study were different solvent proportion (1:2, 1:3 or ST:STUDY_SUMMARY 1:4 v/v) and time under ultrasonic in ice-cold bath (10, 20 or 30 min). The ST:STUDY_SUMMARY extraction solvents were methanol (MeOH) and methyl tert-butyl ether (MTBE). For ST:STUDY_SUMMARY each condition, experiments were prepared in triplicate resulting in 27 samples. ST:STUDY_SUMMARY Samples extracted in MeOH:MTBE (1:2 v/v) for 10 min and 20 min ultrasonic bath ST:STUDY_SUMMARY did not separate in three phases. Then, their organic phase chromatographic ST:STUDY_SUMMARY analyses were not performed resulting on 42 experimental samples chromatograms. ST:STUDY_SUMMARY Ultra-high performance liquid chromatography coupled to electrospray ionization ST:STUDY_SUMMARY mass spectrometry (UHPLC-ESI-MS) was used to acquire raw data and MS-DIAL and ST:STUDY_SUMMARY MetaboAnalyst platforms were used to perform data preprocessing and statistical ST:STUDY_SUMMARY analysis. The statistical analysis of UHPLC-ESI-MS data in both ionization modes ST:STUDY_SUMMARY enabled the visualization of a trend distribution based on extraction time. ST:STUDY_SUMMARY Furthermore, we were able to establish that the solvent proportion had a greater ST:STUDY_SUMMARY impact on group separation in data samples extracted for 30 min versus 10 and 20 ST:STUDY_SUMMARY min. Moreover, diacylglycerol or/and lysophophatidylcholine are lipid subclasses ST:STUDY_SUMMARY that can be favored depending on the extraction time in the MS analyses using ST:STUDY_SUMMARY positive ESI mode. ST:INSTITUTE University of Campinas ST:DEPARTMENT Chemistry's Institute ST:LABORATORY Laboratory of Bioanalytics and Integrated Omics ST:LAST_NAME Matos ST:FIRST_NAME Taynara ST:ADDRESS Rua Josué de Castro, s/n – Cidade Universitária, 13083-970, Campinas – SP, ST:ADDRESS Brazil ST:EMAIL t262827@dac.unicamp.br ST:PHONE (85)996154192 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Pitcairnia flammea #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 - A2_1-POS Time:10 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=A2_1-POS.raw SUBJECT_SAMPLE_FACTORS - A2_2-POS Time:10 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=A2_2-POS.raw SUBJECT_SAMPLE_FACTORS - A2_3-POS Time:10 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=A2_3-POS.raw SUBJECT_SAMPLE_FACTORS - A3_1-POS Time:10 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=A3_1-POS.raw SUBJECT_SAMPLE_FACTORS - A3_2-POS Time:10 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=A3_2-POS.raw SUBJECT_SAMPLE_FACTORS - A3_3-POS Time:10 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=A3_3-POS.raw SUBJECT_SAMPLE_FACTORS - B2_1-POS Time:20 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=B2_1-POS.raw SUBJECT_SAMPLE_FACTORS - B2_2-POS Time:20 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=B2_2-POS.raw SUBJECT_SAMPLE_FACTORS - B2_3-POS Time:20 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=B2_3-POS.raw SUBJECT_SAMPLE_FACTORS - B3_1-POS Time:20 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=B3_1-POS.raw SUBJECT_SAMPLE_FACTORS - B3_2-POS Time:20 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=B3_2-POS.raw SUBJECT_SAMPLE_FACTORS - B3_3-POS Time:20 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=B3_3-POS.raw SUBJECT_SAMPLE_FACTORS - C1_1-POS Time:30 min | Solvent:MeOH;MTBE (1;2 v/v) | Sample_type:Sample RAW_FILE_NAME=C1_1-POS.raw SUBJECT_SAMPLE_FACTORS - C1_2-POS Time:30 min | Solvent:MeOH;MTBE (1;2 v/v) | Sample_type:Sample RAW_FILE_NAME=C1_2-POS.raw SUBJECT_SAMPLE_FACTORS - C1_3-POS Time:30 min | Solvent:MeOH;MTBE (1;2 v/v) | Sample_type:Sample RAW_FILE_NAME=C1_3-POS.raw SUBJECT_SAMPLE_FACTORS - C2_1-POS Time:30 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=C2_1-POS.raw SUBJECT_SAMPLE_FACTORS - C2_2-POS Time:30 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=C2_2-POS.raw SUBJECT_SAMPLE_FACTORS - C2_3-POS Time:30 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=C2_3-POS.raw SUBJECT_SAMPLE_FACTORS - C3_1-POS Time:30 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=C3_1-POS.raw SUBJECT_SAMPLE_FACTORS - C3_2-POS Time:30 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=C3_2-POS_.raw SUBJECT_SAMPLE_FACTORS - C3_3-POS Time:30 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=C3_3-POS.raw SUBJECT_SAMPLE_FACTORS - QC01-POS Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC01-POS.raw SUBJECT_SAMPLE_FACTORS - QC02-POS Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC02-POS.raw SUBJECT_SAMPLE_FACTORS - QC4-POS Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC4-POS.raw SUBJECT_SAMPLE_FACTORS - QC5-POS Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC5-POS.raw SUBJECT_SAMPLE_FACTORS - QC6-POS Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC6-POS.raw SUBJECT_SAMPLE_FACTORS - QC7-POS Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC7-POS.raw SUBJECT_SAMPLE_FACTORS - A2_1-NEG Time:10 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=A2_1-NEG.raw SUBJECT_SAMPLE_FACTORS - A2_2-NEG Time:10 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=A2_2-NEG.raw SUBJECT_SAMPLE_FACTORS - A2_3-NEG Time:10 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=A2_3-NEG.raw SUBJECT_SAMPLE_FACTORS - A3_1-NEG Time:10 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=A3_1-NEG.raw SUBJECT_SAMPLE_FACTORS - A3_2-NEG Time:10 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=A3_2-NEG.raw SUBJECT_SAMPLE_FACTORS - A3_3-NEG Time:10 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=A3_3-NEG.raw SUBJECT_SAMPLE_FACTORS - B2_1-NEG Time:20 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=B2_1-NEG.raw SUBJECT_SAMPLE_FACTORS - B2_2-NEG Time:20 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=B2_2-NEG.raw SUBJECT_SAMPLE_FACTORS - B2_3-NEG Time:20 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=B2_3-NEG.raw SUBJECT_SAMPLE_FACTORS - B3_1-NEG Time:20 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=B3_1-NEG.raw SUBJECT_SAMPLE_FACTORS - B3_2-NEG Time:20 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=B3_2-NEG.raw SUBJECT_SAMPLE_FACTORS - B3_3-NEG Time:20 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=B3_3-NEG.raw SUBJECT_SAMPLE_FACTORS - C1_1-NEG Time:30 min | Solvent:MeOH;MTBE (1;2 v/v) | Sample_type:Sample RAW_FILE_NAME=C1_1-NEG.raw SUBJECT_SAMPLE_FACTORS - C1_2-NEG Time:30 min | Solvent:MeOH;MTBE (1;2 v/v) | Sample_type:Sample RAW_FILE_NAME=C1_2-NEG.raw SUBJECT_SAMPLE_FACTORS - C1_3-NEG Time:30 min | Solvent:MeOH;MTBE (1;2 v/v) | Sample_type:Sample RAW_FILE_NAME=C1_3-NEG.raw SUBJECT_SAMPLE_FACTORS - C2_1-NEG Time:30 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=C2_1-NEG.raw SUBJECT_SAMPLE_FACTORS - C2_2-NEG Time:30 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=C2_2-NEG.raw SUBJECT_SAMPLE_FACTORS - C2_3-NEG Time:30 min | Solvent:MeOH;MTBE (1;3 v/v) | Sample_type:Sample RAW_FILE_NAME=C2_3-NEG.raw SUBJECT_SAMPLE_FACTORS - C3_1-NEG Time:30 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=C3_1-NEG.raw SUBJECT_SAMPLE_FACTORS - C3_2-NEG Time:30 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=C3_2-NEG.raw SUBJECT_SAMPLE_FACTORS - C3_3-NEG Time:30 min | Solvent:MeOH;MTBE (1;4 v/v) | Sample_type:Sample RAW_FILE_NAME=C3_3-NEG.raw SUBJECT_SAMPLE_FACTORS - QC01-NEG Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC01-NEG.raw SUBJECT_SAMPLE_FACTORS - QC02-NEG Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC02-NEG.raw SUBJECT_SAMPLE_FACTORS - QC4-NEG Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC4-NEG.raw SUBJECT_SAMPLE_FACTORS - QC5-NEG Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC5-NEG.raw SUBJECT_SAMPLE_FACTORS - QC6-NEG Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC6-NEG.raw SUBJECT_SAMPLE_FACTORS - QC7-NEG Time:Pooled QC | Solvent:Pooled QC | Sample_type:Quality Control (QC) RAW_FILE_NAME=QC7-NEG.raw #COLLECTION CO:COLLECTION_SUMMARY Leaves of cultivated Pitcairnia flammea were harvested, and immediately frozen CO:COLLECTION_SUMMARY in liquid nitrogen, followed the -80ºC storage until sample preparation. CO:SAMPLE_TYPE Leaves CO:COLLECTION_LOCATION Institute of Biology (UNICAMP, Brazil) #TREATMENT TR:TREATMENT_SUMMARY Leaves were macerated to fine powder using a mortar and pestle in liquid TR:TREATMENT_SUMMARY nitrogen condition. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipid extraction was based on Hummel et al. Method. 10 mL of solvent mixture SP:SAMPLEPREP_SUMMARY were prepared in 3 different proportions - 1:2, 1:3 or 1:4 (v/v) -, pre-cooled SP:SAMPLEPREP_SUMMARY at 20 ºC methanol (MeOH) and methyl tert-butyl ether (MTBE). In 2 mL tubes, 50 SP:SAMPLEPREP_SUMMARY mg of macerated sample were added and 1 mL of the solvent mixture. The samples SP:SAMPLEPREP_SUMMARY were incubated for 5 min under agitation at 500 rpm at 4 ºC, followed by an SP:SAMPLEPREP_SUMMARY ultrasonication in ice-cold bath in different times (10, 20, 30 minutes). Each SP:SAMPLEPREP_SUMMARY condition was performed in technical triplicate, totalizing 27 samples. After SP:SAMPLEPREP_SUMMARY adding 500 µL mixture of water type I:MeOH (3:1 v/v), the samples were vortexed SP:SAMPLEPREP_SUMMARY and centrifuged for 5 min at 4 ºC, 10000 rpm. The three phases were separated SP:SAMPLEPREP_SUMMARY and dried in a vacuum concentrator, at ambient temperature under vacuum - SP:SAMPLEPREP_SUMMARY alcoholic mode, and stored at -80 ºC until the chromatographic analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Scientific UltiMate™ 3000 UHPLC RSLCnano system CH:COLUMN_NAME Supelco Sigma-Aldrich Titan C18 column (100 mm x 2.1 mm x 1.9 µm) CH:SOLVENT_A 40% acetonitrile/60% water; 10 mM ammonium acetate CH:SOLVENT_B 10% acetonitrile/90% isopropanol; 10 mM ammonium acetate CH:FLOW_GRADIENT 250 µL/min: 0-2 min: 40% B, 2-3 min: 50% B, 3-6 min: 50% B, 6.1-8 min: 70% B, CH:FLOW_GRADIENT 8-9 min: 100% B, 9-11 min: 100% B, 11-12 min: 40% B, 12-14 min: 40% B. CH:FLOW_RATE 250 µL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS - MS:MS_RESULTS_FILE ST002570_AN004234_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END