SE58:/MS01
Sample Set Information
ID | SE58 |
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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 raw data files are available at DROP Met web site in PRIMe database of RIKEN.
Analytical Method Details Information
ID | MS01 |
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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. 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 |