SE58:/S01/M01/D01

Sample Set Information

ID	SE58
Title	Exploring matrix effects and quantification performance in metabolomics experiments using artificial biological gradients
Description	We introduce a powerful approach that provides semiquantitative calibration curves over a biologically defined concentration range for all detected compounds. By performing metabolomics on a stepwise gradient between two biological specimens, we obtain a data set where each peak would ideally show a linear dependency on the mixture ratio. An example gradient between extracts of tomato leaf and fruit demonstrates good calibration statistics for a large proportion of the peaks but also highlights cases with strong background-dependent signal interference. Analysis of artificial biological gradients is a general and inexpensive tool for calibration that greatly facilitates data interpretation, quality control and method comparisons.
Authors	Henning Redestig, Makoto Kobayashi, Kazuki Saito, Miyako Kusano
Reference	Henning R et al. (2011) Analytical Chemistry 83: 5645-5651
Comment

The original paper is available at Analytical Chemistry.

The raw data files are available at DROP Met web site in PRIMe database of RIKEN.

Sample Information

ID	S01
Title	Tomato
Organism - Scientific Name	Solanum lycopersicum
Organism - ID	NCBI taxonomy:4081
Compound - ID
Compound - Source
Preparation	Seeds from tomatoes (Solanum lycopersicum, cv. Reiyo) were sown in pots (volume, 2 L) with rockwool (Nittobo, Tokyo, Japan) and grown in a hydroponics system with a nutrient solution containing N, P, and K at 122, 21, and 156.6 mg/L, respectively (Otsuka Chemical, Osaka, Japan), in a growth chamber at 25 ℃/20 ℃ (light/dark) and 900 ppm CO2 concentration with a light/dark cycle of 16 h/8 h at Chiba University, Matsudo, Japan. Photosynthetic photon flux (PPF) level in the growth chamber was adjusted to 450-500 pmol m-2 s-1 when we measured at the meristem of each tomato plant (light source: Ceramic metal halide lamps). Subirrigation was applied twice a day with the nutrient solution, and plant material was harvested three weekdays after flowering in December 2009.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M01
Title	GC-TOF-MS
Method Details ID	MS01
Sample Amount	1 μL(∼5.6 μg of each sample)
Comment

Analytical Method Details Information

ID	MS01
Title	GC-TOF-MS
Instrument	Agilent 6890N gas chromatograph (Agilent Technologies) and Pegasus IV TOF mass spectrometer (LECO)
Instrument Type
Ionization	EI
Ion Mode	Positive
Description	<Extraction and Derivatization for GC-TOF-MS> Each sample was extracted with a concentration of 2.5 mg dry weight (DW) of tissues per ml extraction medium [methanol/chloroform/water (3:1:1 (v/v/v))] containing 10 stable isotope reference compounds: [2H4]-succinic acid, [13C5,15N]-glutamic acid, [2H7]-cholesterol,[13C3]-myristic acid, [13C5]-proline, [13C12]-sucrose, [13C4]-hexadecanoic acid, [2H4]-1,4-butanediamine, [2H6]-2-hydoxybenzoic acid, and 13C6]-glucose. These internal standards were used to normalize the data using cross-contribution compensating mutipl standard normalization (CCMN). Each isotope compound was adjusted to a final concentration of 15 ng/μL for each 1 μL injection. After centrifugation, a 200 μL aliquot of the supernatant (∼0.5 mg of DW of each sample) was drawn and transferred into a glass insert vial for a pilot experiment. We mixed leaf extracts (at the second internode of the second truss) and fruit extracts (mixture of pericarp and jelly/seed) for a gradient experiment. The percentages of leaf:fruit mixture extracts are given in Supporting Information Table 1. The extracts were evaporated to dryness in an SPD2010 SpeedVac concentrator from ThermoSavant (Thermo Electron Corporation, Waltham, MA, USA). For methoximation, 30 μL of methoxyamine hydrochloride (20 mg/mL in pyridine) was added to the sample. After 24 h derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 L MSTFA (Tokyo Chemical Industry, Tokyo, Japan) at 37 ℃ with shaking. A 30 μL aliquot of n-heptane was added following silylation. All derivatization steps were performed in a VSC-100 vacuumglovebox (Sanplatec, Japan) filled with 99.9995% (G3 grade) dry nitrogen. <GC-TOF-MS Conditions> For metabolome analysis, 1 μL of extracts (∼5.6 μg of each sample) was injected in the splitless mode by a CTC CombiPAL autosampler (CTC　Analytics, Zwingen, Switzerland) into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmington, DE, USA) equipped with a 30 m × 0.25 mm inner diameter fused-silica capillary column with a chemically bound 0.25 μL film Rtx-5 Sil MS stationary phase (Restek, Bellefonte, PA, USA). Helium was used as the carrier gas at a constant flow rate of 1 mL min-1. The temperature program for metabolome analysis started with a 2 min isothermal step at 80 ℃ and this was followed by temperature ramping at 30 ℃ to a final temperature of 320 ℃, which was maintained for 3.5 min. The transfer line and the ion source temperatures were 250 and 200 ℃, respectively. Ions were generated by a 70 eV electron beam at an ionization current of 2.0 mA. The acceleration voltage was turned on after a solvent delay of 237 s. Data acquisition was performed on a Pegasus IV TOF mass spectrometer (LECO, St. Joseph, MI, USA) with an acquisition rate of 30 spectra s-1 in the mass rangeof a mass-to-charge ratio of 60 ≦ m/z ≦ 800. Alkane standard mixtures (C8-C20 and C21-C40) were purchased from Sigma-Aldrich (Tokyo, Japan) and were used for calculating the retention index (RI). The normalized response for the calculation of the signal intensity of each metabolite from the mass-detector response was obtained by each selected ion current that was unique in each metabolite MS spectrumto normalize the peak response. For quality control, we injected methylstearate into every sixth sample.
Comment_of_details

Data Analysis Information

ID	D01
Title	Leco ChromaTOF, MATLAB, H-MCR, NIST mass spectral search program
Data Analysis Details ID	DS01
Recommended decimal places of m/z	Default
Comment

Data Analysis Details Information

ID	DS01
Title	Leco ChromaTOF, MATLAB, H-MCR, and NIST mass spectral search program
Description	Nonprocessed MS data from GC-TOFMS analysis were exported in NetCDF format generated by chromatography processing and mass spectral deconvolution software, Leco ChromaTOF version 3.22 (LECO, St. Joseph, MI, USA) to MATLAB 7.0 (Mathworks, Natick, MA, USA), where all data pretreatment procedures, such as smoothing, alignment, time-window setting and H-MCR were carried out. The resolved mass spectra were matched against reference mass spectra using the NIST mass spectral search program for the NIST/EPA/NIH mass spectral library (version 2.0) and our custom software for peak annotation written in Java. Peaks were identified or annotated based on retention indices (RIs) and the reference mass spectra comparison from the Golm Metabolome Database (GMD,http://csbdb.mpimpgolm.mpg.de/csbdb/gmd/msri/gmd_msri.html) released from CSB.DB and our in-house spectral library. The metabolites were identified and defined as annotated metabolites by comparison with RIs from the library databases (GMD and our own library) and with those of authentic standards and mass spectra from these two libraries. Data were normalized using the CCMN algorithm and metabolite identifiers were organized using MetMask.
Comment_of_details