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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14380/m_MTBLS14380_MS_alternating__v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14380/s_MTBLS14380.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14380/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14380/a_MTBLS14380_MS_alternating_.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14380After instrument analysis, raw data were processed using Progenesis QI (Waters Corporation, Milford, USA) for peak detection, extraction, alignment, and integration. Metabolite annotation was performed using the HMDB database (http://www.hmdb.ca/) and the METLIN database (https://metlin.scripps.edu/), as well as the Majji self-built library.To eliminate or reduce errors introduced during experimentation and analysis, the annotated data underwent preprocessing, which included: removal of features with a missing value rate >20% within any experimental group; imputation of remaining missing values with the minimum value across all samples; and normalization of the response intensity of mass spectral peaks across samples using the total sum normalization method, resulting in a normalized data matrix.Subsequently, variables with a relative standard deviation (RSD) >30% in quality control (QC) samples were removed. The data were then subjected to a log10 transformation, yielding the final data matrix for subsequent analyses.Statistical analyses, including PCA and OPLS-DA, were performed using the ropls package (Version 1.6.2) in R. Metabolites were annotated using the HMDB and KEGG (https://www.kegg.jp/kegg/pathway.html) databases. Pathway enrichment analysis was conducted using the scipy.stats package in Python.MetaboLightsPublicMass spectrometry - alternatingUntargeted Metabolomics of Wheat Fielder, TaPDK1-OE, and pdk1 Lines under Alkaline Salt Stress.School of Life Sciences, Shandong UniversityMinghan Cui<54_samples_for_-ms/ms_analysis_protocol>This study included 54 samples for -MS/MS analysis. After instrument analysis, raw data were processed using Progenesis QI (Waters Corporation, Milford, USA) for peak detection, extraction, alignment, and integration. Metabolite annotation was performed using the HMDB database (http://www.hmdb.ca/) and the METLIN database (https://metlin.scripps.edu/), as well as the Majji self-built library.To eliminate or reduce errors introduced during experimentation and analysis, the annotated data underwent preprocessing, which included: removal of features with a missing value rate >20% within any experimental group; imputation of remaining missing values with the minimum value across all samples; and normalization of the response intensity of mass spectral peaks across samples using the total sum normalization method, resulting in a normalized data matrix.Subsequently, variables with a relative standard deviation (RSD) >30% in quality control (QC) samples were removed. The data were then subjected to a log10 transformation, yielding the final data matrix for subsequent analyses.Statistical analyses, including PCA and OPLS-DA, were performed using the ropls package (Version 1.6.2) in R. Metabolites were annotated using the HMDB and KEGG (https://www.kegg.jp/kegg/pathway.html) databases. Pathway enrichment analysis was conducted using the scipy.stats package in Python.Rootmass spectrometry assay1. Sample Preparation and QuenchingFresh wheat root samples were rinsed with ultrapure water to remove soil and debris, then immediately flash-frozen in liquid nitrogen.Frozen roots were ground to a fine powder using a pre-chilled mortar and pestle under continuous liquid nitrogen cooling.Approximately 50 mg of the homogenized powder was accurately weighed into a 2 mL microcentrifuge tube and kept on dry ice or at -80°C until extraction.2. Metabolite Extraction ProcedureThe extraction was performed using a modified methanol/chloroform/water biphasic method optimized for root tissue.Solvent Addition: 800 μL of ice-cold methanol (LC-MS grade, containing internal standards) was added to the powdered tissue. The sample was vortexed vigorously for 30 seconds.Addition of Chloroform: 200 μL of ice-cold chloroform (HPLC grade) was added, followed by another 30 seconds of vortexing.Addition of Water: 400 μL of ice-cold LC-MS grade water was added, and the mixture was vortexed for a final 30 seconds.Incubation: The sample was incubated on ice for 10 minutes to ensure complete metabolite extraction.Centrifugation: The tube was centrifuged at 14,000 × g for 15 minutes at 4°C to separate the phases.3. Sample Collection and Preparation for LC-MSThe upper aqueous phase (containing polar metabolites) was carefully transferred to a new microcentrifuge tube.The extract was dried completely using a vacuum concentrator (CentriVap, 35°C).The dried metabolites were reconstituted in 100 μL of 50% methanol (v/v, in water) with 0.1% formic acid.The reconstituted sample was vortexed for 30 seconds, sonicated in an ice-water bath for 5 minutes, and then centrifuged at 14,000 × g for 10 minutes at 4°C to remove insoluble debris.The final supernatant was transferred to an LC-MS vial for analysis.4. Quality Control (QC) and Control SamplesPooled QC Sample: An aliquot (e.g., 10 μL) from each individual sample extract was combined to create a pooled QC sample. This QC sample was injected at the beginning of the analytical run and after every 5-10 experimental samples to monitor instrument stability and performance.Solvent Blank: A blank sample containing only the extraction solvents (methanol, chloroform, water) and internal standards was processed identically to the biological samples to account for background contamination.Internal Standards: A mixture of stable isotope-labeled compounds (e.g., leucine-13C6, phenylalanine-13C9, 2H4-succinic acid) was spiked into the initial methanol solvent prior to extraction to correct for variations in extraction efficiency and matrix effects.5. Additional NotesAll steps were performed on ice or at 4°C where possible to minimize metabolite degradation.The lower organic phase (chloroform layer) can be collected separately for lipidomics analysis if required.The use of acidified reconstitution solvent (0.1% formic acid) is optional and depends on the LC-MS method (typically beneficial for positive ionization mode in LC-ESI-MS).Triticum aestivumhttps://www.ebi.ac.uk/metabolights/MTBLS14380Minghan Cui. Shandong University. cuiminghande@163.com.After instrument analysis, raw data were processed using Progenesis QI (Waters Corporation, Milford, USA) for peak detection, extraction, alignment, and integration. Metabolite annotation was performed using the HMDB database (http://www.hmdb.ca/) and the METLIN database (https://metlin.scripps.edu/), as well as the Majji self-built library.To eliminate or reduce errors introduced during experimentation and analysis, the annotated data underwent preprocessing, which included: removal of features with a missing value rate >20% within any experimental group; imputation of remaining missing values with the minimum value across all samples; and normalization of the response intensity of mass spectral peaks across samples using the total sum normalization method, resulting in a normalized data matrix.Subsequently, variables with a relative standard deviation (RSD) >30% in quality control (QC) samples were removed. The data were then subjected to a log10 transformation, yielding the final data matrix for subsequent analyses.Statistical analyses, including PCA and OPLS-DA, were performed using the ropls package (Version 1.6.2) in R. Metabolites were annotated using the HMDB and KEGG (https://www.kegg.jp/kegg/pathway.html) databases. Pathway enrichment analysis was conducted using the scipy.stats package in Python.Alkalicuiminghande@163.comFielder, TaPDK1-OE, and pdk1 were grown under normal conditions until the two-leaf stage. Fielder was designated as JW1, and pdk1 as PDK1-KO. The control group continued to be cultured under normal conditions. For the D3 group, the culture solution was replaced with one containing 50 mM alkaline salt and cultured for three days. For the D1 group, the culture solution was replaced with one containing 50 mM alkaline salt and cultured for one day, with sampling performed at the respective time points.MetabolomicsMetabolomicsRootuntargeted analysisThermo Scientific Q Exactive HF-XOpenMSexperimental blankShandong UniversityTriticum aestivumuntargeted metabolite profilingMetabolomicsRootuntargeted analysisThermo Scientific Q Exactive HF-XOpenMSexperimental blankShandong UniversityTriticum aestivumuntargeted metabolite profilinginstrument  Thermo Scientific Q Exactive HF-Xion source ESIionisation mode positive & negativem/z range 70-1050falseUntargeted Metabolomics of Wheat Fielder, TaPDK1-OE, and pdk1 Lines under Alkaline Salt StressAn untargeted metabolomics study was performed on wheat plants, including the wild-type cultivar Fielder, TaPDK1-overexpression (TaPDK1-OE) lines, and pdk1 mutant lines. Plants were grown under either normal conditions (control groups) or exposed to alkaline salt stress for 1 or 3 days (experimental groups). Each treatment group consisted of six independent biological replicates.2026-04-262026-04-26MTBLS14380