CIMR n/a http://msi-workgroup.sourceforge.net/ Taken direct from Metabolomics vol. 3(3), 2007 (PDF) on 2011-06-06 by Chris Taylor (chrisftaylor@gmail.com). Sections renumbered throughout to begin at 1. Mammalian and other in vivo experiments The experiments considered include functional genomic studies, drug toxicology, nutrigenomics, clinical trials, and other human studies. Two reporting requirements are described for pre-clinical (e.g. functional genomics, toxicology) and clinical (e.g. clinical trials, nutrigenomics) studies. Note that this version corrects the layout errors in the published version by using the previous draft from http://msi-workgroups.sf.net/ as a reference. 1 Standards for mammalian pre-clinical studies This pre-clinical category includes many animal experiments in functional genomics, drug discovery, and early stage drug safety assessment where the experimental system can be closely controlled within a laboratory environment. 1.1 Experimental subject description Species or strain designation For rat or mouse http://www.informatics.jax.org/mgihome/nomen/strains.shtml shall Generation of mixed strain If different from [base] species and strain as given. While ‘generation of mixed strains’ is a requested field, a number of physiological studies have found a profound influence on the phenotype of an animal during that study as a result of drift in the strain background. should Model description Surgical, pharmacological or feeding manipulation shall Animal supplier Company, location, colony designation if any, if wild caught shall Age range Dates of birth, as well as ages at time of experiment shall Weight range Give individual weights if available shall 1.2 Husbandry 1.2.1 Housing While many of these terms are optional, an important potential source of biological variation in many drug safety assessment studies relying on the use of metabolism cages (metabowls) to collect urine is the stress associated with changing the environment of an animal. Many mice and rats display profound changes in their urinary profiles during the first 24 h in a new environment. Group or individual shall Cage type shoe box, metabolic, wire mesh should Cage change or cleaning frequency should Environmental enrichment should 1.2.2 Light cycle shall 1.2.3 Feed Type and manufacturer Or reference to composition if custom diet. shall Ad lib or restricted diet 25 grams per day shall Diet supplements If any (what treats, how often, how much) should 1.2.4 Water Bottle or automated shall Tap or purified Qualified distilled, 18 MΩ shall 1.2.2 Veterinary treatments if any and exercise regimen (large animals) should 1.2.6 Use of anesthesia For example, for blood collection or physicals. [N.B. Numbering absent in manuscript -- corrected.] should Type of anesthetic Formulation, time of administration, dose of anesthetic should 1.2.7 Acclimation Acclimation duration To experimental facility should Acclimation duration To diet (if experimental diet differs) should 1.3 Experimental design 1.3.1 Number of groups Numbers, gender and group sizes shall 1.3.2 Inclusion criteria physical exams, normal metabolomic model screen should 1.3.3 Treatments Compound shall Route shall Dose shall Dose volume shall Duration of dosing shall Vehicle shall 1.3.4 Fasting When relative to metabonomic sample collection. shall Duration of fast in hours should 1.3.5 End Points Euthanasia method shall Tissue collection list shall Tissue processing method snap freezing shall Clinical signs Time of observation relative to dose. shall Body weights and food consumption How often measured should Blood chemistries, haematology, histopathology, special assays The terms blood chemistry, haematology, histology and special assays may be populated in many studies, but these terms certainly will not be universal, especially in laboratories with limited resources, hence they were not deemed to be required information. should 1.4 Metabolomics-related sample collection 1.4.1 Blood The metabolic profiles of serum and plasma are markedly different, and there is still much work required to define what metabolites are removed during the clotting procedure involved in serum production. In addition the use of anticoagulants and other additives will also affect the resultant biological matrix and have an impact on the final results produced. Volume collected shall Location of collection shall Time of collection Relative to dose and light cycle. shall Serum or plasma Give anticoagulant or presence of serum separator. shall Storage conditions shall Temperature shall Duration should 1.4.2 Urine How collected metabolic cage, cystocentesis, catheterisation shall Frequency of collection shall Duration of collection shall Time of collection relative to dose and light cycle If less than 24 hours shall Bacteriostatic agent or any other additive final concentration shall Urine volume For 24 hour collections should Temperature of urine collection tube On ice/room temp should Storage conditions shall Temperature shall Duration should 1.4.3 Tissues The metabolic profiles of serum and plasma are markedly different, and there is still much work required to define what metabolites are removed during the clotting procedure involved in serum production. In addition the use of anticoagulants and other additives will also affect the resultant biological matrix and have an impact on the final results produced. Identification shall Approximate quantity taken shall Tissue processing method snap freezing, time from kill to snap freezing shall Storage conditions shall Temperature shall Duration should 2 Standards for mammalian clinical trials and human studies The 'clinical' category includes experiments largely involving human subjects where changes are monitored in a less controlled environment such as occurs in clinical trials, nutritional studies, epidemiological investigations and samples examined from tissue banks. 2.1 Experimental subject description Ethical approval details shall Geographical location If subjects are at a hospital please identify it. shall Ethnic background Based on FDA and Office of National statistics criteria. should Demographics should Medical history Disease or clinical symptoms; criteria for disease presence (all volunteers should not have factors in their medical history which confound the study); e.g. surgical, pharmacological manipulation, medication (may be referenced). shall Age range shall Weight range and height May provide BMI as well or instead. shall Gender shall Trial type randomised trial, disease biomarker, phase I-IV shall Diet and dietary restrictions Describe relevant control groups for such dietary restrictions. Diet - standardized, isocaloric, free living subjects. if applicable shall Further descriptors smoking, blood pressure, anomalies in habitual diet (e.g. vegetarian, vegan etc.), sporting activity and frequency, habitual alcohol consumption. shall 2.2 Experimental design 2.2.1 Number of groups Numbers, gender, group size shall 2.2.2 Inclusion criteria shall 2.2.3 Exclusion criteria shall 2.2.4 Treatment and fasting Compound shall Route shall Dose shall Dose volume shall Duration of dosing shall Vehicle shall 2.2.5 End points Clinical chemistries, blood chemistry and haematology urea, creatinine, glucose, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, total protein, albumin, erythrocyte count, haemoglobin, haemocrit, platelets, white blood count, sodium, potassium, bilirubin, ALT, ALP, -GT. should Urine chemistry osmolality, ketones, pH, protein, glucose, bilirubin, blood, sediment and colour. should 2.3 Metabolomics-related sample collection 2.3.1 Blood The metabolic profiles of serum and plasma are markedly different, and there is still much work required to define what metabolites are removed during the clotting procedure involved in serum production. In addition the use of anticoagulants and other additives will also affect the resultant biological matrix and have an impact on the final results produced. Volume collected shall Location of collection shall Serum or plasma Identify any anticoagulant; (serum only), time allowed for clotting and temperature; (plasma and serum), temperature of centrifugation, time and speed of centrifugation. shall Arterial or venous blood collected should Observations of haemolysis In samples, with reporting of whether samples were used in subsequent analysis. shall Time from separation to freezing/freezing process should Storage conditions shall Temperature shall Duration should 2.3.2 Urine Frequency and volume of collection State whether mid-flow or total urine. shall Bacteriostatic agent or any other additive final concentration shall Storage conditions shall Temperature shall Duration should 2.3.3 Tissues The metabolic profiles of serum and plasma are markedly different, and there is still much work required to define what metabolites are removed during the clotting procedure involved in serum production. In addition the use of anticoagulants and other additives will also affect the resultant biological matrix and have an impact on the final results produced. Identification shall Approximate quantity taken shall Post mortem tissues Hours after death until sample collection shall Storage conditions shall Temperature shall Duration should Microbial and in vitro biology experiments -- sample source This section seen by the authors as 'best practice' rather than 'minimal'. N.B., The last point of the penultimate section ('Harvesting'), and all of the final section ('Biotic fators related to sample work-up'), which refer to the [CFT-renamed] 'sample processing' sister document, have been omitted. Experimental design Biological question or the goal of the experiment Experimental design details Preferably in the form of a diagram or table, showing relationships between the following. Samples Treatments or growth conditions Time or timing of sample collection Extracts Repeats Metabolome samples Phenotypic characteristics of the samples Other 'omics' data sets generated from these same samples Biosource Source or supplier of the cell line or strain ATCC In case of natural isolates If at all possible, cultures should be deposited in an international culture collection with an accession number and relevant details. What taxonomic system was used to identify the (micro-)organism? Because micro-organisms are often identified incorrectly. In case of mutant strains From which wild-type were they obtained and how? In case of (higher) eukaryotes Cell type, organ derivation, grade of differentiation, subcellular location Immortalized or transformed If applicable shall Cell storage Growth environment Growth container: Type, supplier, geometry of the fermenter/bioreactor, (shake) flask or microtiter plates Growth supports (type and supplier) in case of cells cultured in adherence. Growth configuration suspension or attached culture, monolayer, double layer, sandwich, spheroids, batch, fed-batch, perfusion, continuous fermentation Inoculation procedure Subculturing and splitting protocols Inoculation size, seeding density Inoculation size, seeding density (volume percentage [v.v^-1], num of cells per ml for suspension cultures; num of cells per cm^2 for cells in adherence, subconfluence or confluence. Medium or substrates Type and supplier, concentration or percentage -- including additions and supplementations (antibiotics, growth factors, serum type, and batches, concentration or percentage). Environmental conditions Which of the environmental conditions were controlled and which could alter (freely) during growth? Temperature, pH, gas composition, humidity, percent CO2, stirrer speed, evaporation, pO2 Growth rate If the cells were grown at a set fixed growth rate: which was the growth rate? Treatment and incubation conditions Treatment factors Biotic competition with or infection by other organisms Abiotic physical stresses, chemical substances treatment dose and vehicle Treatment time and intervals Labeling protocols for any use of or incubation with labeled substrates 13C compounds used (percent enrichment, purity) if applicable shall Pretreatment if applicable shall Harvesting Biotic characteristics of moment of harvesting growth phase/stage (logarithmic, stationary, steady state, cytostatic phase, cell cycle phase, ...), number of generations in case of continuous cultures, time of sampling, stabilization time/phase before experiment, number of culture passages, independent indicators of differentiated state (immunological or molecular markers) Abiotic characteristics at time of harvesting cell density [OD, DWT, counts], depletion of nutrients, treatment time Phenotypic characteristics especially relating to the question under study yield, productivity, color, form Microbial and in vitro biology experiments -- sample processing Sampling What is the time between sample removal from its environment until metabolic activity is truly stopped? What was the temperature during this process? How were the samples harvested? Quenching How was the metabolism of the samples shut down? How is the cell integrity under the quenching conditions? Extracellular metabolites How were intracellular metabolites discriminated from extracellular metabolites? Extraction of metabolites from the cells How were the (intracellular) metabolites extracted from the cells? What is the estimated recovery at this step? Normalization of the metabolome data How were the metabolome data normalized? Specifically (in case of normalizing the data with respect to the amount of cells [no., mg] that they were obtained from), how and at what step was the amount of biomass determined? Sample clean-up or work-up How were the samples cleaned-up with respect to compounds that interfere with analysis? Sample storage How and how long were the samples stored after collection, during work-up and prior to analysis? Quality control steps How was it verified that no biotic or abiotic changes occurred, or were at least minimized, during the complete sample collection and work-up phase? Detection limit What is the detection limit of the metabolites for the samples analyzed in the study? Stability What is known about the stability of (specific) metabolites during quenching, extraction and sample preprocessing? Environmental context N.B., Sections numbered from the section titles rather than the document (i.e., E3.2 not 8.4.4) S Sample S1 Description of the biological sample involved in the study Taxonomic classification of organism(s) Please give details of all organisms sampled, as far along the taxonomic scale as possible, ideally to the levels of genus, species and sub-species. Refer to a taxonomic classification, such as the NCBI taxonomy (http://www.ncbi.nlm.nih.gov/Taxonomy/) or the Integrated Taxonomic Information System (http://www.itis.gov/) Common name(s) vernacular where possible shall Genotype(s) where possible shall Ecotypes where possible shall Sample composition Please provide details of the organism(s) that constitute the sample. Amounts may be described in ‘absolute’ (number of individuals) or relative terms (50% Organism X; 25% Organism Y; 25% Unknown). Sample Type Please give details of the type of sample (for example; Community, Population, Whole Organism, Organ, Biofluid, etc.). Condition of specimen(s) Please give details of general observations on health, etc. should Phenotypic characteristic(s) should Weight of specimen(s) should Age(s) of specimen(s) should Sex(es) of specimen(s) should Stage(s) of development should Image data. Please provide copies of any photographs of samples taken in the field during collection (or URLs to such images). should E Environment E1 Description of ANY field environment Geographic location Please specify latitude and longitude in decimal degrees. If relevant, you can also provide position in a local coordinate system e.g. the UK’s Ordnance Survey grid (http://www.ordnancesurvey.co.uk/) Altitude or depth Please specify in meters above/below sea level. Habitat Please provide a descriptor of habitat type. Meteorological conditions weather type (sunny, snowing, etc.), humidity, precipitation, wind speed and direction should Lunar or solar phase should All other measured parameters Pollutant concentration(s) E2 Description of ANY laboratory environment Please refer to the MSI—In vivo context requirements (Griffin et al.) and the MSI Plant context requirements (Nikolau et al.). Laboratory address and contact details E3.1 Description of terrestrial environment See also the requirements in E1, E2. Inclination and aspect Substrate type Substrate temperature All other measured parameters substrate pH, substrate organic content should E3.2 Description of aquatic environment See also the requirements in E1, E2. Sample(s) was submerged and emerged (how deep and for how long in this condition) Water temperature Tidal phase should All other measured parameters pH, salinity, dissolved (in)organic content should E3.3 Description of atmospheric environment See also the requirements in E1, E2. Atmospheric temperature All other measured parameters atmospheric pressure, (in)organic content should E3.4 Description of biotic environment See also the requirements in E1, E2. An organism can have a 'biotic environment', where the immediate environment of an organism is another organism (for example in a parasitic relationship). This scenario necessitates a nested description of the environment, including a description of the 'biotic environment' of the primary organism and that of the host. Description of host organism Relationship of organism(s) to host parasite All other measured parameters pH, temperature should P Process P1 Description of capture or sampling of sample or organism(s) Description of capture or sampling procedure Include details such the method used netted, electrically stunned, anaesthetised, razor cut Reason for capture should Other capture parameters should P2 Description of storage or preservation of sample(s) Description of storage/preservation procedure Include details such as the storage or preservation medium liquid nitrogen, dry ice, formaldehyde Reason for storage/preservation should Other storage or preservation parameters temperature should P3 Description of maintenance of organism(s) Please also refer to the MSI—In vivo context requirements (Griffin et al.) and the MSI Plant context requirements (Nikolau et al.). Description of maintenance procedure Include details such as the type of housing, for example; Cage, aquaria, continuous culture, seed bag or plant pot, etc. Reason for maintenance should Other maintenance parameters feeding regime, cage dimensions should P4 Description of transportation of samples or organism(s) Transportation may involve storage or maintenance; see also the requirements in either P2 or P3 as appropriate. Description of transportation procedure Include details such as the means of transport, for example; refrigerated container. Reason for transportation should Other transportation parameters should P5 Description of acclimation of organism(s) Acclimation involves maintenance; see also the requirements in P3 as appropriate. Description of acclimation procedure Include details such as the type of housing, for example; cage, aquaria, continuous culture, seed bag or plant pot, etc. Reason for acclimation should Other acclimation parameters should P6 Description of general manipulation of sample or organism(s) Record details of any controlled manipulation as part of the study. Manipulation type Perturbation exposure to a toxicant, dissection, sacrifice Reason for manipulation should Description of manipulation procedure perturbation of specific environmental parameter, dissection of specific tissue Other manipulation parameters should Plant biology context Sections renumbered from 3.1 - 3.4 to 1 - 4. 1 BioSource This term refers to the physical objects that were subjected to metabolomic analyses, consisting of a description of the species and genotype and the organ that was sampled, and the bulk quantity of sample. In certain cases, more detailed information may be available such as organ specifications, cell types or subcellular compartments. These metadata are only required and meaningful if sampling methods that allowed such annotations were used. The methods used for sampling should be named in such cases to enable independent evaluation of data. Species Names of species should be described according to the NCBI taxonomy database (Wheeler et al. 2000; Benson et al. 2000) (http://pubmedexpress.nih.gov/Taxonomy/taxonomyhome.html/index.cgi). Plant species need to be named in full and not abbreviated, e.g. Arabidopsis thaliana. All necessary information on taxonomic relationships can be derived from the correct species name and thus does not need to be reported further. Genotype Subspecies information such as ecotype, cultivar and accession should be described according to authoritative databases such as TAIR (http://www.arabidopsis.org/). In the case of crosses or breeding results, available pedigree information must be given. In the case of transgenic or mutant organisms, name of the gene(s) that are up- or down-regulated should be reported, and the GenBank Accession number(s) for the sequence(s) of the corresponding construct(s), in addition to the parental subspecies background information, should be given. According to standard practice in agronomic genotype nomenclature, genotype description should comprise the author who first described or collected the cultivar, e.g. Medicago truncatula (Gaertn) cv. Jemalong. If available, registrations numbers for agronomic plants should be referenced, e.g. USDA GRIN. The number of backcrosses used in breeding needs to be detailed. In case of plant–pathogen interaction studies or other studies where information on multiple genomes is relevant, such metadata should be given. Organ Names of organs and plant structure should be described according to the authoritative database (Katica et al. 2007) maintained by the Plant Ontology Consortium to be found at http://www.plantontology.org/. All necessary information on organ relationships can be derived from the correct organ name and thus does not need to be reported further. Organ specification This should be provided only if such information cannot be detailed by http://www.plantontology.org/ (e.g. description of a part of an organ, the specific location of the organ or a specific tissue of an organ). Cell type This should be provided only if such information can be detailed in a meaningful manner, e.g. by cell sorting or dissection. Naming according to the authoritative database maintained by the Plant Ontology Consortium is to be found at http://www.plantontology.org/ under plant_structure ontology. Only if such information cannot be located at this source the Cell Ontology maintained at Open Biomedical Ontologies group should be taken, which is to be found at http://lists.sourceforge.net/lists/listinfo/obo-cell-type. Subcellular location This should be described only if such information can be detailed in a meaningful manner, e.g. by subcellular fractionation. Naming according to the authoritative database (Gene Ontology Cellular Component) maintained by the Gene Ontology Consortium to be found at http://www.geneontology.org/ BioSource amount This refers to the mass (mg fresh weight or mg dry weight), number of cells or other measurable bulk quantities (e.g. protein content). 2 Growth environment This section specifically excludes variation of growth conditions that were part of the experimental design, i.e. factors that were altered with the intention to cause metabolic differences. Such differences in study parameters should be reported as ‘treatment’. Although it cannot be made mandatory, documentation of additional metadata should be regarded as part of best plant biology practice, such as application of agrochemicals or biotic plant protection. When investigating other documents relating to the specifics of plant growth reporting requirements, a document was retrieved that was published by the International Committee for Controlled Environment Guidelines (http://ncr101.montana.edu/) in March 2004, detailing the ‘Minimum Guidelines for Measuring and Reporting Environmental Parameters for Experiments on Plants in Growth Rooms and Chambers’ (http://ncr101.montana.edu/min_guidelines.pdf). While we appreciated the efforts of this committee, we felt that many of the recommendations were rather demanding to be put into practice in current laboratory settings, especially in public research institutions. The intention of documents detailing minimal reporting standards, including the paper presented here, is to detail enough information to re-use data and to understand the concepts and layout of experimental designs. If minimal requirements ask for a level of detail that is usually not reported by researchers, these can hardly serve as consensus, which would be endorsed and followed by the majority of active investigators. Instead, we suggest such guidelines to be part of ‘best practice’ documents. Growth support Soil (type, supplier), Agar (type, supplier), Vermiculite (type, supplier), hydroponic system (type, supplier, nutrient concentrations) or other support including cell culture (media, volume, cell number per volume). Growth location Field trial (location), climate chamber (size m^3), greenhouse (details on accuracy of control of light, humidity and temperature conditions), other location (details on size m^3, accuracy of control of light, humidity and temperature conditions). Growth plot design The way to randomize the different genotype-environment interactions. Either descriptive or using established nomenclature e.g. latin square. Light Light quality, light source model/type, light intensity (best reported as empirically measured at plant height), luminescence (daylight) period (h). For field trials: average light parameters in growing season. Information on time and location of the field trial enables tracking of more precise information if necessary. Humidity Humidity (percentage) at day and at night. For field trials: average humidity parameters in growing season. Information on time and location of the field trial enables tracking of more precise information if necessary. Temperature Temperature (°C) at day and at night. For field trials: average temperature (°C) at day and at night in growing season. Information about time and location of the field trial enables tracking of more precise information if necessary. Watering regime Amount and time of watering per day. For field trials: average rain fall in growing season. Information on time and location of the field trial enables tracking of more precise information if necessary. For hydroponic systems: frequency of solution change. Nutritional regime Amount and time of additional nutrients given to plants. Date(s) of plant establishment Depending on plant study, such dates could comprise: sowing, germination, transplanting, cutting, grafting or other appropriate time stamps. Plant development stage description should accompany time stamps using established nomenclature (Boyes et al. 2001; Pujar et al. 2006; Palmquist et al. 2006). Other specific metadata translocation of plants from one chamber to another, rotational schema of trays within a climate chamber, agrochemical or preventive maintenance information that is not part of 'treatment' factors. 3 Treatment Treatment factors Biotic treatment infection (species), herbivore attack (species), competition with other plants (species) Abiotic treatment light intensity variations, cold acclimation (temperature), heat stress, drought (description of residual growth support moisture, or quantitative description of reduction in watering regime), water stress, saline stress Intervention treatment application of agrochemicals, enzyme inhibitors, hormones, elicitors Treatment dose or intensity levels Depending on treatment factors. Treatment time, time intervals and duration before harvest Depending on treatment factors and treatment time. 4 Harvest The harvest determines the set point for stopping metabolism, analogous to sampling time points in related documents on biology context metadata. Harvest date and time Harvest time relative to the luminescence cycle. Duration of harvest if relevant to the plant study (e.g. for volatile analysis). Plant growth stage It is advised to refer to established literature, e.g. for Arabidopsis (Boyes et al. 2001) and Medicago truncatula (Palmquist et al. 2006); and for general growth stage ontology (Pujar et al. 2006). Time after harvest before stopping cellular metabolism May be greater than weeks for certain post-harvest physiology experiments, may be less than seconds for assessing high turnover metabolites. Metabolism quenching method Method to stop cellular metabolism Harvest method Details of operation to gather the plant organ (sample) Sample storage Details of pooling of plant tissues for analysis; operations to store sample (e.g. freeze-drying, grinding) prior to preparation for metabolomic analysis; duration and temperature of storage before extraction for analysis. Chemical analysis Sections renumbered from 2.1 - 2.10 to 1 - 10. 1 Sample preparation Sampling process and protocol Replicate sampling and analyses Substantial biological variance exists within all organisms; therefore replicate sampling and analyses are critical to provide a statistical basis for data evaluation and interpretation. A minimum of triplicate (n = 3) biological sampling is proposed with n = 5 preferred. Biological replicates (repetitive analyses of samples obtained from different individuals or pooled individuals from a population) are preferred over analytical replicates (repetitive analyses of the same sample obtained from the same individual or pooled individuals) as biological variance almost always exceeds analytical variance. Tissue harvesting method For example, sample freezing method (e.g. liquid N2, dry ice and acetone bath, freeze clamping, etc.), sample wash method for removing unwanted external components, time and duration for tissue collection (e.g. time from tissue resection to liquid N2 freezing), temperature, and sample storage prior to further preparation (e.g. –80 degrees celsius for 2 weeks). All temperatures should be measured if possible; however temperature setpoints are acceptable assuming quality monitoring was performed and no abnormalities recorded. Biofluid harvesting or collection method For example, syringe, collection onto refrigerated surface, vacuum system/vacutainers used for blood collection, storage vessel and anticoagulant (if relevant), temperature, velocity and duration of centrifugation, and sample freezing method. Tissue processing method For example, lyophilization, fresh tissue processing, pulverization/homogenization, tissue cell lysis (e.g. liquid N2 grinding, manual or electric homogenization, beadbased homogenization, ultrasonic cell lysis, buffer based lysis, etc.). Storage conditions prior to extraction or further processing –80°C, duration, atmospheric pressure or vacuum, desiccation, preservatives added Relocation and shipping of tissues from one laboratory to another Extraction method Solvent(s), pH and ionic strength of buffer, solvent temperature and volume(s) per quantity of tissue, number of replicate extracts, sequential extraction, and extraction time. It is noted that degassing of solvents is important to minimize redox reactions of sensitive compounds such as ascorbate, cysteine, etc. 1 ml ice-cold methanol (MeOH) per 6 mg lyophilized tissue, two extractions combined, CHCl3/MeOH (2/1, v/v) followed by 10% trichloroacetic acid extraction. Extract concentration, dilution, and resolubilization processes Dried under nitrogen, resolubilized in H2O or pyridine Extract Enrichment if relevant shall Solid phase extraction Column volume/mass, elutant, sorbent, manufacturer. Desalting, molecular weight cut-off, ion exchange, etc. Extract cleanup and additional manipulation ultrafiltration, removal of paramagnetic ions, addition of metal chelators such as EDTA, citrate Extract storage and relocation Storage conditions prior to and during analysis Relocation and shipping of extracts from one laboratory to another if relevant shall 2 Chromatography Chromatography instrument description Manufacturer, model number, software package and version number or date. Auto-injector Injector model/type, software version, injection volume, wash cycles (volumes), solvent. Separation column and pre- or guard column Manufacturer, model number/name, stationary media composition (support and coating, e.g. silica, C18, etc.) and physical parameters (i.e. coating thickness for GC/MS, particle size and pore size for LC/MS), internal diameter, and length. Technique-specific sample preparation Resuspension of sample (e.g. in mobile phase), amount injected. Derivatization reaction conditions if relevant, (e.g. OMS/trimethylsilyl; chemical manufacturer, temperatures, and duration). Sample spiking e.g. internal standards, retention index standards. Separation parameters Method name (a detailed method can be published elsewhere and referenced here by a unique protocol identifier), injector temperature, split or splitless mode and ratio, LC post-column split, mobile phase compositions, mobile phase flow rates, pressure, thermal/solvent/solute gradient profiles. 3 Mass spectrometry Instrument description Manufacturer, model number, software package and version (name, number or date). Sample introduction and delivery From GC, from LC, direct infusion without chromatography, direct infusion using dedicated autosampler flow rate. Ionization source Ionization mode (EI, APCI, ESI etc.), polarity (positive or negative-ion analysis), vacuum pressure, skimmer/focusing lens voltages (e.g. capillary voltage etc.), gas flows (e.g. nebulization gas, cone gas etc., source temperature). Although these values will vary between instruments, they should provide a cumulative view of the ionization conditions sufficient to enable reproduction of the experiment. Mass analyzer description and acquisition mode Type (quadrupole, ion-trap, time-of-flight, FT-ICR, including combinations of these for hybrid instruments), acquisition mode (full scan, MSn, SIM, MRM, etc.). Technique-specific sample preparation if relevant shall Re-suspension of sample (e.g. in MeOH:water 1:1 with 0.2% formic acid), derivatization, volume injected, and internal calibrant(s) added (if relevant). Data acquisition parameters Date, operator, data acquisition rate, m/z scan range, compounds used for m/z calibration, mass resolution, mass accuracy, logic program used for data acquisition (often reported for ion-traps), spectral acquisition rate, vacuum pressure, and/or lock spray (concentration, lock mass, flow rate, and frequency). 4 Nuclear magnetic resonance spectroscopy Instrument description Manufacturer, model name/number, magnetic field strength in Tesla (example 14.1 T Varian Inova; 18.8 T Bruker Avance) or proton resonance frequency e.g. 600 MHz, and console description. Instrument configuration VT control, pulsed field gradients (z or x,y,z) and maximum gradient strength (if used), number of shims, number of channels. Probe type (e.g. 10 mm 31P, 5 mm HCN cold probe, 3 mm flow-probe etc.), solution or solid-state, automation or manual operation, autotune or manual tune, and probe gas. For LC-NMR: sample handler, injection volumes, wash cycles and solvent. Instrument-specific sample preparation Volume, extract/powder/intact organisms, tissue or cells, type of NMR tube (e.g. conventional, Shigemi, microcell etc.), pH, solvent (D2O, CD3OD, CDCl3 etc.), buffer, chemical shift or calibration standard. Data acquisition parameters For 1-D 1H or X-nucleus NMR: temperature, observed nucleus, pulse sequence name, pulse sequence implementation (e.g. gradient selection, sensitivity enhancement), spin rate or statement of no spin, solvent saturation or decoupling method, presence or absence of heteronuclear decoupling (e.g. isotope-enriched samples), decoupling mode and bandwidth; spin lock field strength (in Hz) and duration (in sec), mixing time (for NOESY, ROESY etc.), spin echo time (e.g. for relaxation analysis or broadline suppression), RF pulse widths, any selective pulse shapes and durations used, magnetic field gradient pulse times and shapes, spectral width, acquisition time, relaxation delay and additional delays (mixing time, etc.), interpulse delay (or recycle time), digitization parameters, spectral width and acquisition time, number of transients, and number of steady states transients (i.e. dummy scans). For solvent suppression: technique, excitation maximum and bandwidth. Additional parameters for 2-D and higher dimensional NMR: observed nucleus in F2 and F1, pulse sequence, excitation pulse widths for relevant nuclei, spectral width in F2 and F1, solvent saturation method, number of transients in t2 and number of increments in t1, acquisition times for t2 and t1, phase sensitive or magnitude detection. pulsed field gradient strengths and shapes (z or x,y,z) and maximum gradient strength (if relevant to the pulse sequence). Additional parameters for X-nucleus 1D and higher dimensional NMR: direct or indirect detection, proton decoupling mode (Waltz, Garp, Wurst, Stud etc.) and effective band width, evolution time for constant time experiments, editing mode (cf. INEPT-based experiments), heteronuclear spin lock strength and mixing time (e.g. HCCH-TOCSY). Additional parameters for pseudo 2D NMR experiments: physical parameter varied in the t1 dimension (e.g. T2, T1, diffusion period, chromatographic separation time as in LC-NMR, etc.), pulse sequence, array of values used for physical constants. 5 stable isotopes & flux analysis Isotope labeled precursors used Element/isotope, position(s), percent labeled; e.g. [13C-1]-D-glucose (98%), [15N2]-L-glutamine (99%). Isotope source (i.e. manufacturer), chemical purity of the labeled compound(s), concentration of the compound, fraction of total present (requires detailed breakdown of media composition for cell and tissue studies, including analysis of any added FCS or other growth supplements; labeling scheme). Total number of moles isotope added during the experiment. Duration of pulse label or continuous addition 6 Fourier transform infrared (FT-IR) spectroscopy FT-IR spectrometer instrument description Manufacturer, model number, software name and version number or date. Instrument configuration Type of sampling compartment used, including where necessary type of microscope employed. Type of detector used (DTGS (deuterated triglycine sulphate), MCT (mercury cadmium telluride), and/ or FPA (focal plane array). Technique-specific sample preparation Resuspension of n mg ml–1 sample into solvent, volume analysed. Sample presentation Transmission measurement: in KBr, or on ZnSe, Si windows. Reflectance measurement: on Si, Au, Al, or other defined metal sample carrier. Diffuse reflectance measurement: on defined metal sample carrier. Sampling area, and for imaging pixel size. Data acquisition parameters Wavenumber (cm–1) range. Rate of acquisition. Spectral resolution (in cm^–1). Number of spectra co-added. Number of data points in the resultant spectrum, and how this is displayed (absorbance or transmission). 7 Instrumental performance and method validation Minimum Reporting of Instrumental Performance Parameters is Encouraged. The nature and method(s) used to ensure sensitive and selective instrumental performance should be reported and the following details and descriptors are deemed appropriate. Mass spectrometry instrument performance validation parameters reported might include chemical description of the m/z calibration standard used, accuracy of m/z calibration, mass resolution, and ion source optimization parameters. For hyphenated MS methods, suggested reporting parameters could include chromatographic resolution, accuracy and precision of internal standard(s) or retention time markers, accuracy and precision for replicated analyses, accuracy and precision for validation sample(s), and cycles per column/injector/septum/ blank. NMR instrument performance verification parameters might include calibration standard used (name, chemical shift and concentration; e.g. 0.5 mM DSS or 1 mM TMS at 0.0 ppm), statement of line width of the standard at 50% and 1% of its full height (e.g. DSS, TSP or TMS methyl peak) or residual water, pH marker used (if relevant) and shift correction. For X nuclei: external or internal reference and conditions, and correction made for susceptibility effects. Reporting of shift referencing method for indirect dimension in 2D experiments (direct or indirect based on c ratios) would also be beneficial. Quantitative Method Validation. Two methods of quantitative analysis are typically used in metabolomics and include relative and absolute quantification. Relative quantification (i.e. reporting of metabolite(s) instrument response relative to an internal standard or another metabolite(s) level such as the sum of all metabolite abundance) is typically used in non biased metabolomics. Whereas, absolute quantification (determination of the absolute concentration of a metabolite(s) through correlation of its instrument response to that of a known concentration series of the same metabolite) is commonly used in targeted metabolite(s) analysis. Relative Quantification A description and quantifier of the added exongenous isotopically labeled or unlabeled metabolite(s). A description of the method used for assessing instrument response (e.g. peak integration, binning/ bucketing or deconvolution method, intensity normalized to reference, For NMR, descriptions for correction for saturation effects - T1 values measured), and provide relaxation agents if added (type, amount). For direct X-detection (especially 13C or 31P), correction for nuclear Overhauser enhancement as well as saturation. For non-deuterated aqueous samples, state any corrections made for nonlinear excitation profile and method. Reporting on replicate analyses, standard error/ deviation of quantification. Absolute Quantification Calibration curves should be generated for each metabolite to be quantified in the same biological matrix and include a sufficient number of standard solutions to adequately define the instrument response to concentration relationship (i.e. suggested minimum of at least one standard solution per order of change in concentration). The range of standard solutions used and the range of linearity with correlation coefficient should be reported. A quantifier of the method accuracy (i.e. standard deviation, relative standard deviation, coefficient of variance) should be reported and bias assessed if possible (bias; due to method, lab, ion suppression, etc.). A quantifier of the method precision (i.e. standard deviation, relative standard deviation, coefficient of variance) should be reported. The lower limit of quantification (LLOQ) and confidence level should be reported. The LLOQ is defined as the minimum concentration generating an instrumental signal-to-noise response ratio of 10. The LLOQ has alternatively been defined as 5 times the limit of detection (LOD). The LOD is defined as the concentration that yields a minimum instrumental signal-to-noise ratio of 3. Additional quantitative descriptions of recovery and/or stability provide additional method validation. 8 Data preprocessing Post Acquisition Data Pre-processing Data file format used and/or conversion methods should be reported. Examples include conversion of proprietary file formats to more universal formats such as net.cdf, XML, MZmine, etc. Details of any data pre-processing methods which convert raw instrumental data into organized or tabular file formats should be reported. Examples for MS might include: background subtraction, noise reduction, curve resolution for temporal chromatographic alignment, peak picking, peak thresholding, spectral deconvolution, and/or metabolite identifications. Some comparative methods do not resolve or identify individual metabolites prior to comparative analysis. The general experimental details describing these methods should still be reported and should be sufficient so that others can replicate the data processing. Examples for NMR data pre-processing might include phase-correction method (e.g. automatic, manual), conversion from time to frequency domain (e.g. Fourier Transform), degree of zero filling, degree of linear prediction; apodization parameters and window functions in all dimensions (exponential, Gaussian, sine bell etc.), baseline corrections (dc offset, linear or non-linear corrections), first point multipliers, any shifting of the free induction decays. For data analysis of isotope labeling of flux experiments, the method for determining positional and fractional labeling, standard error of the estimates; and estimated isotope recovery in observable fractions (and fraction of total isotope supplied) should be described. Examples for FT-IR spectroscopy might include conversion from time to frequency domain (e.g. Fourier Transform), and degree of zero filling. Baseline corrections parameters might include offsets, level and type of derivatisation (including algorithm, window size for smoothing), and whether or not CO2 was removed from spectra (deleted or a linear trend fitted). 9 Identification of metabolites Non-novel metabolites A minimum of two independent and orthogonal data relative to an authentic compound analyzed under identical experimental conditions are proposed as necessary to validate non-novel metabolite identifications (e.g. retention time/index and mass spectrum, retention time and NMR spectrum, accurate mass and tandem MS, accurate mass and isotope pattern, full 1H and/or 13C NMR, 2-D NMR spectra). The use of literature values reported for authentic samples by other laboratories are generally believed insufficient to validate a confident and rigorous identification. The use of literature or external laboratory data result in level 2 identifications. If spectral (MS or NMR) matching is utilized in the identification process then the authentic spectra used for the spectral matching should be described appropriately or libraries made publicly available. It is preferred that the reference spectra are made available at no cost, but the CAWG recognizes that this may not always be possible for commercialized libraries (NIST, Wiley, etc.). However, the premise of this minimum is that authors document and provide the spectral evidence to validate the metabolite identifications. If the authors choose not to provide the experimental evidence to support the identifications, then the identifications should be reported as ‘putative identifications’. Metabolite identifications based upon additional orthogonal data (i.e. more than two) are highly advantageous, provide additional confidence, and are often necessary to provide unambiguous identification of stereo configuration. Additional data consistent with best chemical practices might include: selective solvent extraction, retention time, m/z, photodiode array spectra, kmax and emax,chemical derivatization, isotope labeling, 2D NMR, IR spectra, etc. Novel metabolites Describe extraction, isolation, and purification followed by elemental analysis, accurate mass measurement, ion mass fragmentation patterns, NMR (1H, 13C, 2D), and other spectral data such as IR, UV, or chemical derivatization. Unknown metabolites N.B., originally numbered as section 10 (2.10 in original). For NMR, the exact chemical shift and multiplicity of at least one nucleus in the metabolite should be part of the unknown nomenclature For example, an unidentified triplet at 1.16 ppm could be reported as: ‘unknown (1.16 ppm, triplet)’. When such a signal can be correlated with other atoms in the same molecule using multidimensional or multi-pulse techniques, the chemical shifts and the connectivity of such correlated nuclei in the unknown should be reported in the work. In such cases the molecular fragement may be identified, such as ‘isopropyl group’. For MS, the retention time, retention index, and/or prominent ions in the mass spectrum should be reported along with MS-MS data if available (also see Bino et al. 2004). Xenobiotics (e.g. administered drugs, related drug metabolites) or other exogenous compounds such as herbicides, pesticides, etc. should be rigorously distinguished from endogenous metabolites for all unknowns and if possible. NMR spectroscopy This is the 'ABC' group's minority report on NMR included in CIMR (and the group publication) despite the overlap with the CAWG Sample description Reference to biological sample provenance details pH of biological sample should pH of sample after buffer has been added Field frequency lock Chemical name Additional solute For example, buffers or chelating agents should Chemical name Concentration in sample Solvent One or more. Chemical name Concentration in sample Chemical shift standard should Chemical name Concentration in sample Concentration standard should Chemical name Internal or external Concentration in sample Instrument description Geographical location of the instrument Magnet Serial number should Manufacturer Model Field strength Probe Serial number should Manufacturer Model Gradient strength Console Serial number should Manufacturer Model Acquisition computer Serial number should Manufacturer Model Operating system and version number Application software and version number Autosampler Serial number should Manufacturer Model Application software and version number Acquisition parameters Parameters recorded once for each sample Acquisition parameters file reference Shaped pulse file reference 0..* Sample details Sample introduction method tube, flow probe, rotor Size of tube or flow probe or rotor Sample temperature in autosampler should Sample temperature in magnet Instrument operation details Sample spinning rate Water suppression technique Pulse sequence name Pulse sequence file reference Pulse sequence literature reference Data acquisition details Number of steady-state scans Number of scans Relaxation delay Parameters recorded once for each NMR analysis dimension Instrument operation details Irradiation frequency Acquisition nucleus 90° pulse width of irraditation nucleus Data acquisition details Dwell time Number of data points acquired Parameters recorded for higher dimensions Name of encoding scheme Hadamard frequency 0..* Quality control Identity of signal within output data Linewidth of signal prior to window function processing Full width at half-maximum (FWHM) Peak width at five percent total height FID and spectral processing parameters Parameters recorded once when a raw data set is processed Post-acquisition water suppression method name should Time-based to frequency-based data transformation method Name of chemical used to reference the spectrum Processing software details 1..* Processing software name and version number Spectral projection details 0..* Method of projection Projection axis Parameters recorded once for each NMR analysis dimension Processing parameters file reference should Number of data points in spectrum Zero order phase correction should First order phase correction should Calibration reference shift Baseline correction method should Spectral de-noising method should Window function details Window function name Function parameter and value 1..* Parameters recorded for the second dimension only 2D J-resolved processing details 0..1 Flag to indicate if the data has been tilted Flag to indicate if the data has been symmetrised Spectral quantitation parameter set Quantitation type Quantitation algorithm Quantitation parameters should Manual quantitation description should Processing software details 1..* Processing software name and version number Analysis description Date and time of data acquisition Institution Operator Supervisor