ONTOLOGY SOURCE REFERENCE
Term Source Name	"NCBITAXON"	"CHMO"	"EFO"	"ROLEO"	"NCIT"	"OBI"	"CHEBI"	"MS"	"CHEMINF"	"BTO"
Term Source File	"http://data.bioontology.org/ontologies/NCBITAXON"	"http://data.bioontology.org/ontologies/CHMO"	"http://data.bioontology.org/ontologies/EFO"	"http://data.bioontology.org/ontologies/ROLEO"	"http://data.bioontology.org/ontologies/NCIT"	"http://data.bioontology.org/ontologies/OBI"	"http://data.bioontology.org/ontologies/CHEBI"	"http://data.bioontology.org/ontologies/MS"	"http://data.bioontology.org/ontologies/CHEMINF"	"http://data.bioontology.org/ontologies/BTO"
Term Source Version	"2"	"18"	"146"	"1"	"44"	"22"	"99"	"86"	"41"	"29"
Term Source Description	"National Center for Biotechnology Information (NCBI) Organismal Classification"	"Chemical Methods Ontology"	"Experimental Factor Ontology"	"Role Ontology"	"National Cancer Institute Thesaurus"	"Ontology for Biomedical Investigations"	"Chemical Entities of Biological Interest Ontology"	"Mass Spectrometry Ontology"	"Chemical Information Ontology"	"BRENDA Tissue and Enzyme Source Ontology"
INVESTIGATION
Investigation Identifier	"MTBLS66"
Investigation Title	"Investigation"
Investigation Description	""
Investigation Submission Date	"2017-09-05"
Investigation Public Release Date	"2019-03-01"
Comment[Created With Configuration]	"C:tempISAcreatorMetaboLightsConfigurationsMetaboLightsConfig20150707"
Comment[Last Opened With Configuration]	"MetaboLightsConfig20150707"
INVESTIGATION PUBLICATIONS
Investigation PubMed ID	""
Investigation Publication DOI	""
Investigation Publication Author List	""
Investigation Publication Title	""
Investigation Publication Status	""
Investigation Publication Status Term Accession Number	""
Investigation Publication Status Term Source REF	""
INVESTIGATION CONTACTS
Investigation Person Last Name	""
Investigation Person First Name	""
Investigation Person Mid Initials	""
Investigation Person Email	""
Investigation Person Phone	""
Investigation Person Fax	""
Investigation Person Address	""
Investigation Person Affiliation	""
Investigation Person Roles	""
Investigation Person Roles Term Accession Number	""
Investigation Person Roles Term Source REF	""
STUDY
Study Identifier	"MTBLS66"
Study Title	"A large-scale metabolomics study to harness chemical diversity and explore biochemical mechanisms in ryegrass (UPLC-MS C1 reverse phase POS and GC-MS FAME assays)"
Study Description	"Perennial ryegrass (<i>Lolium perenne</i>) is integral to temperate pastoral agriculture, which contributes most of the milk and meat production worldwide. Chemical profiles and diversity of ryegrass offer several opportunities to harness specific traits and elucidate underlying biological mechanisms for forage improvement. We conducted a large-scale metabolomics study of perennial ryegrass comprising 715 genotypes, representing 118 populations from 21 countries. Liquid/gas chromatography-mass spectrometry based targeted and non-targeted techniques were used to analyse fructan oligosaccharides, lipids, fatty acid methyl esters, polar and semi-polar compounds. Fructan diversity across all genotypes was evaluated, high- and low-sugar groups identified, and fructan accumulation mechanisms explored. Metabolites differentiating the two groups were characterised, modules and pathways they represent deduced, and finally, visualisation and interpretation provided in a biological context. We also demonstrate a workflow for large-scale metabolomics studies from raw data through to statistical and pathway analysis. <br><br> <b>UPLC-MS C1 reverse phase POS and GC-MS FAME assays</b> are reported in the current study MTBLS66.<br> <b>UPLC-MS HILIC POS assay</b> is reported in <a href=https://www.ebi.ac.uk/metabolights/MTBLS62>MTBLS62</a>.<br> <b>UPLC-MS HILIC NEG assay</b> is reported in <a href=https://www.ebi.ac.uk/metabolights/MTBLS63>MTBLS63</a>.<br> <b>UPLC-MS C18 reverse phase POS assay</b> is reported in <a href=https://www.ebi.ac.uk/metabolights/MTBLS64>MTBLS64</a>.<br> <b>UPLC-MS C18 reverse phase NEG assay</b> is reported in <a href=https://www.ebi.ac.uk/metabolights/MTBLS65>MTBLS65</a>.<br> <b>UPLC-MS C1 reverse phase NEG assay</b> is reported in <a href=https://www.ebi.ac.uk/metabolights/MTBLS68>MTBLS68</a>.<br> <b>HPLC-MS Hypercarb reverse phase NEG assay</b> is reported in <a href=https://www.ebi.ac.uk/metabolights/MTBLS529>MTBLS529</a>.<br>"
Study Submission Date	"2017-09-05"
Study Public Release Date	"2019-03-01"
Study File Name	"s_MTBLS66.txt"
STUDY DESIGN DESCRIPTORS
Study Design Type	"reversed-phase chromatography"	"ultra-performance liquid chromatography-mass spectrometry"	"untargeted metabolites"	"Lipidomics"	"fatty acid methyl ester"	"gas chromatography-mass spectrometry"	"targeted metabolites"
Study Design Type Term Accession Number	"http://purl.obolibrary.org/obo/CHMO_0002302"	"http://purl.obolibrary.org/obo/CHMO_0000715"	""	"http://ncicb.nci.nih.gov/xml/owl/EVS/Thesaurus.owl#C153193"	"http://purl.obolibrary.org/obo/CHEBI_4986"	"http://purl.obolibrary.org/obo/CHMO_0000497"	""
Study Design Type Term Source REF	"CHMO"	"CHMO"	""	"NCIT"	"CHEBI"	"CHMO"	""
STUDY PUBLICATIONS
Study PubMed ID	""
Study Publication DOI	"10.1038/s42003-019-0289-6"
Study Publication Author List	"Arvind K. Subbaraj, Jan Huege, Karl Fraser, Mingshu Cao, Susanne Rasmussen, Marty Faville, Scott J. Harrison and Chris S. Jones"
Study Publication Title	"A large-scale metabolomics study to harness chemical diversity and explore biochemical mechanisms in ryegrass"
Study Publication Status	"In preparation"
Study Publication Status Term Accession Number	""
Study Publication Status Term Source REF	""
STUDY FACTORS
Study Factor Name	"Genotype"	"Replicate"	"Country of origin"	"Population ID"	"Type"	"MFFGC accession no."
Study Factor Type	"germline genotype"	"biological replicate"	"geographic location"	"population"	"cultivar"	"Accession Number"
Study Factor Type Term Accession Number	"http://www.ebi.ac.uk/efo/EFO_0004973"	"http://www.ebi.ac.uk/efo/EFO_0002091"	"http://purl.obolibrary.org/obo/GAZ_00000448"	"http://purl.obolibrary.org/obo/OBI_0000181"	"http://www.ebi.ac.uk/efo/EFO_0005136"	"http://ncicb.nci.nih.gov/xml/owl/EVS/Thesaurus.owl#C25402"
Study Factor Type Term Source REF	"EFO"	"EFO"	"CHEMINF"	"EFO"	"EFO"	"NCIT"
STUDY ASSAYS
Study Assay File Name	"a_MTBLS66_LC_metabolite_profiling_mass_spectrometry.txt"	"a_MTBLS66_GC_metabolite_profiling_mass_spectrometry.txt"
Study Assay Measurement Type	"metabolite profiling"	"metabolite profiling"
Study Assay Measurement Type Term Accession Number	"http://purl.obolibrary.org/obo/OBI_0000366"	"http://purl.obolibrary.org/obo/OBI_0000366"
Study Assay Measurement Type Term Source REF	"OBI"	"OBI"
Study Assay Technology Type	"mass spectrometry"	"mass spectrometry"
Study Assay Technology Type Term Accession Number	"http://purl.obolibrary.org/obo/OBI_0000470"	"http://purl.obolibrary.org/obo/OBI_0000470"
Study Assay Technology Type Term Source REF	"OBI"	"OBI"
Study Assay Technology Platform	"Exactive (Thermo Scientific)"	"DSQ II Trace Ultra (Thermo Scientific)"
STUDY PROTOCOLS
Study Protocol Name	"Sample collection"	"Extraction"	"Chromatography"	"Mass spectrometry"	"Data transformation"	"Metabolite identification"
Study Protocol Type	"Sample collection"	"Extraction"	"Chromatography"	"Mass spectrometry"	"Data transformation"	"Metabolite identification"
Study Protocol Type Term Accession Number	""	""	""	""	""	""
Study Protocol Type Term Source REF	""	""	""	""	""	""
Study Protocol Description	"Ryegrass seeds were obtained from the Margot Forde Germplasm Centre at AgResearch Limited, Grasslands Research Centre, Palmerston North, New Zealand. Seeds of 724 genotypes (denoted by a specific code e.g. PG0698) which included NZ cultivars (82), natural population/ecotypes (49), NZ elite breeding populations (336), overseas cultivars (181), enhanced germplasm (71) and unknown cultivars (5) were selected. 5 clonal replicates (n = 5; denoted as PG0698_1 for the first replicate) per genotype were cultivated and used for metabolomics studies."	"At 60 days after transplanting, leaves were harvested during late autumn (May 2012), snap-frozen in liquid nitrogen, freeze-dried, ground to a coarse powder, transferred to glass vials and stored at -80 °C until further use. 5 aliquots of 50 mg each (4 for analyses and 1 standby) of each sample were weighed into microcentrifuge tubes. A surrogate QC sample59 comprising random amounts of ground ryegrass leaf material, irrespective of the presence or absence of endophyte was used. Since this QC sample was not representative of the sample set, it was solely used for monitoring sample degradation and for tracking run-order effects within a batch, and not used for batch normalisations or ensuing data processing. </br></br> Boiling water (1.5 ml) was added to another aliquot (50 mg). After homogenisation, samples were placed in a hot water bath (90 °C) for 30 min. Samples were cooled to room temperature, centrifuged, and approximately 500 µl of the aqueous extract was transferred to an autosampler vial for analysis. Samples were stored (-20 °C) for a minimal period to avoid fructan degradation. After extraction of fructans, the remaining supernatant was discarded and the pellet was freeze-dried overnight. The dried aliquot was transferred to a 15 ml Falcon tube containing 50 µl of pentadecanoic acid (C15:0; CAS No. 1002-84-2; MW = 242.4) in heptane (4 mg/ml) as an internal standard. After the addition of 1 ml of 1 M methanolic HCl reagent, the tubes were purged with nitrogen, sealed and heated in a hot water bath (80 °C) for 1 hr. Tubes were then cooled to room temperature, and 50 µl of Heptadecanoic acid methyl ester (Premethyl ester C17:0; CAS No. 1731-92-6; MW = 284.48) in heptane (4 mg/ml) was added as another internal standard. 600 µl of heptane and 1 ml of 0.9% sodium chloride (w/v; NaCl) were added and the tubes were manually shaken to extract Fatty Acid Methyl Esters (FAMEs) into the heptane phase. 150 µl of the heptane layer was transferred to a 250 µl glass insert fitted in an autosampler vial for GC-MS analysis. </br></br> A single aliquot (50 mg) was used for analysing lipid compounds. 1 ml of extraction solvent comprising damp chloroform:methanol (67:33, v/v) and containing 2′,7′-Dichlorofluorescein (20 µg/ml) as an internal standard, were added to the aliquot. A ceramic bead was added and samples were mixed in a bead-mill homogeniser (TissueLyser II; Qiagen, Valencia, CA, USA) for 3 min, after which they were centrifuged for 6 min (18188 x g). Approximately 500 µl of the aqueous extract was transferred to an autosampler vial and stored at -20 °C until analysis."	"The Thermo LC–MS system (Thermo Fisher Scientific, Waltham, MA, USA) for analysis of lipids consisted of an Accela 1250 quaternary UHPLC pump, a PAL auto-sampler fitted with a 15,000 psi injection valve (CTC Analytics AG., Zwingen, Switzerland) a 20 μl injection loop, and a Q-Exactive Orbitrap mass spectrometer with electrospray ionisation. A Betasil C1 column (50 x 2.1 mm, 5 µm; Thermo Fisher Scientific, Waltham, MA, USA), maintained at 25 °C with a gradient elution program and a flow rate of 500 µl/min, was used for chromatographic separation. The mobile phase comprised water containing 0.1% formic acid (Solvent A) and isopropanol:acetonitrile containing 0.1% formic acid (50:50, v/v; Solvent B). The gradient was set to hold solvent A at 80% from 0-1 mins, gradually decline to 0% at 18.1 mins, maintained at the same level up to 20 mins, increased to 80% at 20.1 mins and finally allowed to equilibrate as such for the rest of the program, i.e. 25 mins. The samples were cooled in the auto-sampler at 4 °C and the injection volume of each sample was 2 μl. The first 1.5 min and the last 5 min of the chromatogram were diverted to waste. </br></br> Analysis of FAMEs was undertaken using a Thermo DSQ II Trace Ultra gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) fitted with a DB-5 GC (1 µm, 0.25 mm x 15 m; Agilent Technologies) capillary column. GC-MS conditions were as described previously[1]. Briefly, the parameters are: Acquisition Time 20 min; Source Temp 200 °C; and Start Time 10 min. </br></br> For each stream of analysis, samples (3575) were systematically randomised across 36 batches, making sure that 2 clonal replicates of a genotype were not present in the same batch. A single batch comprised approximately 100 samples interspersed with a QC sample for every 10 samples (10-12 QC samples per batch). The sequence comprised blanks, QC and samples. </br></br> Ref:</br> [1] Browse J, McCourt PJ, Somerville CR. Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem. 1986 Jan;152(1):141-5. PMID:3954036</br>"	"For LC-MS, both full and data dependent MS2 scans were collected in profile data acquisition mode. For full scan mode, a mass resolution setting of 35,000 was set to record a mass range of m/z 200–2000 with a maximum trap fill time of 250 ms. For MS2 scan mode, the same mass resolution setting was maintained with a maximum trap fill time of 120 ms. The isolation window of selected MS1 scans was ± 1.5 m/z with a collision energy of 30 eV. Samples were run in positive ionisation mode. Positive ion mode parameters were as follows: spray voltage, 4.0 kV; capillary temperature, 275 °C; capillary voltage, 90 V, tube lens 120 V. The nitrogen source gas desolvation settings were (arbitrary units): sheath gas, 40; auxiliary gas, 10; sweep gas, 5. The Xcalibur software package provided by the manufacturer was used to create these settings. </br></br> For GC-MS, data was collected on a Thermo Scientific DSQ II with a mass range of m/z 50-350."	"Collected data were processed using the Xcalibur software package provided by the manufacturer."	"XCMS[1] and in-house R scripts with appropriate parameters for Lipids settings were used for: 1) peak detection using centwave; 2) grouping; 3) retention time alignment using obiwarp; 4) peak re-grouping; and 5) filling of missing peaks. </br></br> The resultant data matrices were cleaned and post-processed by using: 1) diffreport function of XCMS to generate extracted ion chromatograms of all identified peaks, and eliminating the ones that represented background noise, and; 2) CAMERA[2] to identify and eliminate isotopes (HILIC and C18), and 3) normalisation for batch-effects using comBat[3]. The final data matrices of metabolic features were used for local library matching and statistical analysis. Metabolic features, defined here as molecular entities or ion types with a unique mass-to-charge ratio (m/z) and retention time. </br></br> Lipid identification was performed using LipidSearch software (Thermo Fisher Scientific, USA)[4]. Raw lipid files corresponding to high- and low-sugar groups were uploaded to LipidSearch separately for positive ionisation mode. Product ion search on Q-Exactive data was selected with a mass tolerance of 6 ppm for precursor ions and 10 ppm for product ions, along with a selection of lipid classes (see Table S4 in the paper associated with this study). Lipid species/classes identified for each file were then merged based on retention time alignments and a single file with the merged results, 1 each for each ionisation mode, was generated. This library of identified lipids was matched against the final data matrices based on parent mass and retention times. Peak intensities generated by XCMS settings for the identified lipids were ultimately used for further statistical analyses. </br></br> Ref:</br> [1] Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G. XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem. 2006 Feb 1;78(3):779-87. doi:10.1021/ac051437y. PMID:16448051</br> [2] Kuhl C, Tautenhahn R, Böttcher C, Larson TR, Neumann S. CAMERA: an integrated strategy for compound spectra extraction and annotation of liquid chromatography/mass spectrometry data sets. Anal Chem. 2012 Jan 3;84(1):283-9. doi:10.1021/ac202450g. PMID:22111785</br> [3] Johnson WE, Li C, Rabinovic A. Adjusting batch effects in microarray expression data using empirical Bayes methods. doi:10.1093/biostatistics/kxj037. PMID:16632515</br> [4] Peake DA, Yokoi Y, Wang J, Huang Y. A new lipid software workflow for processing orbitrap-based global lipidomics data in translational and systems biology research. THERMO SCIENTIFIC application note (2013).</br>"
Study Protocol URI	""	""	""	""	""	""
Study Protocol Version	""	""	""	""	""	""
Study Protocol Parameters Name	""	"Post Extraction;Derivatization"	"Chromatography Instrument;Autosampler model;Column model;Column type"	"Scan polarity;Scan m/z range;Instrument;Mass analyzer;Ion source"	""	""
Study Protocol Parameters Name Term Accession Number	""	";"	";;;"	";;;;"	""	""
Study Protocol Parameters Name Term Source REF	""	";"	";;;"	";;;;"	""	""
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STUDY CONTACTS
Study Person Last Name	"Subbaraj"
Study Person First Name	"Arvind"
Study Person Mid Initials	"K."
Study Person Email	"Arvind.Subbaraj@agresearch.co.nz"
Study Person Phone	"+6463518060"
Study Person Fax	""
Study Person Address	"Grasslands Research Centre, Tennent Drive, Palmerston North, New Zealand 4442"
Study Person Affiliation	"AgResearch Ltd."
Study Person Roles	"scientist"
Study Person Roles Term Accession Number	"http://purl.obolibrary.org/obo/RoleO_0000477"
Study Person Roles Term Source REF	"ROLEO"
