Profiling of Arabidopsis secondary metabolites by capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry

Abstract

Large-scale metabolic profiling is expected to develop into an integral part of functional genomics and systems biology. The metabolome of a cell or an organism is chemically highly complex. Therefore, comprehensive biochemical phenotyping requires a multitude of analytical techniques. Here, we describe a profiling approach that combines separation by capillary liquid chromatography with the high resolution, high sensitivity, and high mass accuracy of quadrupole time-of-flight mass spectrometry. About 2000 different mass signals can be detected in extracts of Arabidopsis roots and leaves. Many of these originate from Arabidopsis secondary metabolites. Detection based on retention times and exact masses is robust and reproducible. The dynamic range is sufficient for the quantification of metabolites. Assessment of the reproducibility of the analysis showed that biological variability exceeds technical variability. Tools were optimized or established for the automatic data deconvolution and data processing. Subtle differences between samples can be detected as tested with the chalcone synthase deficient tt4 mutant. The accuracy of time-of-flight mass analysis allows to calculate elemental compositions and to tentatively identify metabolites. In-source fragmentation and tandem mass spectrometry can be used to gain structural information. This approach has the potential to significantly contribute to establishing the metabolome of Arabidopsis and other model systems. The principles of separation and mass analysis of this technique, together with its sensitivity and resolving power, greatly expand the range of metabolic profiling.

Figure 1.

Manual deconvolution of data generated by CapLC-ESI-QqTOF-MS. A 53-min LC-MS analysis generated a total ion chromatogram consisting of 1,590 single MS spectra. A, The total ion chromatogram was manually deconvoluted in 25-D slices resulting in single extracted ion chromatograms (XICs) covering, for example, a mass range from 200 to 225 D. B, Mass spectra were then extracted from peaks apparent within a particular time frame of the XIC. The extracted peak at t_R 26.0 min (C, left) represents sinapoyl malate ([M+Na]⁺ m/z 363.0717;[2 sinapoyl malate+K]⁺ m/z 719.1148; in-source fragment at m/z 207.0658). The extracted XIC valley at 26.6 min (C, right) represents a typical background mass spectrum.

Figure 2.

Technical and biological variance of the CapLC-ESI-QqTOF-MS analysis assessed by analyzing 25 mass signals from leaf extracts and 22 mass signals from root extracts. Signals are identified by mass and t_R. Technical reproducibility of the LC-MS analysis of methanolic root (A) and leaf extracts (C) was determined by extracting the same material three times as well as performing three repetitions of the LC-MS measurements. Quantification of 22 selective ion traces in root extracts and 25 ion traces in leaf extracts was carried out. The sd of the three independent LC-MS analyses of a single extract is represented in the white, gray, and black bars of the charts. The cumulative sd for repeated analysis of the same extract showed a value of 11.1%. Comparison of the three different extracts of the same material (striped bar) resulted in an average variability of 25% ± 17.9% for leaves and 21.6% ± 13.4% for roots. When four independent experiments were analyzed, an average biological variation was measured of 35.5% ± 14.0% for leaf signals (D) and 55.9% ± 26.0% for root signals (B).

Figure 3.

Mass spectra of two metabolites differentially accumulating in Arabidopsis wild-type (Ler WT) and mutant plants (Ler tt4 mutant). The flavonoid glycoside Rha-Rha-kaempferide ([M+H]⁺ at m/z 579.1731, in-source fragments at m/z 433.1107 and 287.0500) was not detected in leaves of the tt4 mutant line (A), whereas the glucosinolate gluconasturtiin ([M+Na]⁺ at m/z 446.0611) accumulated to much higher amounts in the roots of tt4 plants (B).

Figure 4.

Identification of significant metabolites of Arabidopsis by HR-ESI-MS/MS (product ion scan). A, Positive ion CID-MS of gluconasturtiin (in the box: calculated and measured isotopic pattern of the [M+Na]⁺ ion at m/z 446). B, Positive ion ESI-CID-MS of hirsutin.