13 CO 2 labeling. The heavy carbon of
13 CO 2 is quickly incorporated
in various primary metabolites in photosynthesizing leaves [5]. In
species, which emit isoprene from leaves, rapid
13 C labeling of
isoprene via the plastidic methylerythritol phosphate (MEP) pathway is closely linked to photosynthetic CO 2 fixation in the CalvinBenson cycle [6, 7]. Part of carbon fluxes through these pathways
also maintain the levels of photosynthetic pigments (carotenes and
chlorophyll a) which undergo continuous turnover in illuminated
leaves [8]. Insights into dynamic pathway regulation and interaction in different genotypes and environments can be gained by
quantitative analysis of isotope incorporation in metabolites of the
corresponding pathways.
Mass spectrometry (MS) can reliably differentiate isotopically
labeled metabolites based on their mass. Accurate mass determination by high resolution MS, such as Fourier transform ion cyclotron
resonance (FTICR)-MS and Orbitrap-MS, enables unambiguous
annotation of metabolites by empirical formula calculation
[9]. Today MS-based technologies coupled with separation techniques, such as liquid chromatography (LC) and gas chromatography, have become an essential platform to study metabolic
pathways. Especially, they provide powerful tools for nontargeted
or multitargeted metabolite analysis (metabolite profiling and fingerprinting) in complex biological samples, thus complementing
the fast advancement in genomic, transcriptomic and proteomic
approaches [9–11]. When combined with stable isotope labeling,
metabolite snapshots obtained from a time course experiment can
provide dynamic information about pathway fluxes in a metabolic
network [12, 13].
Here we describe a method for carotenoid profiling in
13
Clabeled leaf extracts using LC- MS and LC-FTICR-MS. Pigments
are extracted from leaves of plants that are cultivated in a
13
CO 2
environment [14, 15]. After separation by LC and detection by
(photo)diode array detector (PDA or DAD), ionization is performed by electrospray ionization (ESI) or atmospheric pressure
chemical ionization (APCI) in the positive mode [16–18]. Mass
assignment of product ions to specific carotenoid isotopologs is
achieved by FTICR-MS. Based on peak intensity, the relative abundance and the degree of
13
C labeling are calculated for individual
carotenoid isotopologs.
2 Materials
Prepare all aqueous solutions with ultrapure deionized water and
analytical grade (LC-MS grade) reagents. Discard remaining
extracts and solvents following waste disposal regulations.
264
Bjo ¨ rn Thiele and Shizue Matsubara
13 CO 2 is quickly incorporated
in various primary metabolites in photosynthesizing leaves [5]. In
species, which emit isoprene from leaves, rapid
13 C labeling of
isoprene via the plastidic methylerythritol phosphate (MEP) pathway is closely linked to photosynthetic CO 2 fixation in the CalvinBenson cycle [6, 7]. Part of carbon fluxes through these pathways
also maintain the levels of photosynthetic pigments (carotenes and
chlorophyll a) which undergo continuous turnover in illuminated
leaves [8]. Insights into dynamic pathway regulation and interaction in different genotypes and environments can be gained by
quantitative analysis of isotope incorporation in metabolites of the
corresponding pathways.
Mass spectrometry (MS) can reliably differentiate isotopically
labeled metabolites based on their mass. Accurate mass determination by high resolution MS, such as Fourier transform ion cyclotron
resonance (FTICR)-MS and Orbitrap-MS, enables unambiguous
annotation of metabolites by empirical formula calculation
[9]. Today MS-based technologies coupled with separation techniques, such as liquid chromatography (LC) and gas chromatography, have become an essential platform to study metabolic
pathways. Especially, they provide powerful tools for nontargeted
or multitargeted metabolite analysis (metabolite profiling and fingerprinting) in complex biological samples, thus complementing
the fast advancement in genomic, transcriptomic and proteomic
approaches [9–11]. When combined with stable isotope labeling,
metabolite snapshots obtained from a time course experiment can
provide dynamic information about pathway fluxes in a metabolic
network [12, 13].
Here we describe a method for carotenoid profiling in
13
Clabeled leaf extracts using LC- MS and LC-FTICR-MS. Pigments
are extracted from leaves of plants that are cultivated in a
13
CO 2
environment [14, 15]. After separation by LC and detection by
(photo)diode array detector (PDA or DAD), ionization is performed by electrospray ionization (ESI) or atmospheric pressure
chemical ionization (APCI) in the positive mode [16–18]. Mass
assignment of product ions to specific carotenoid isotopologs is
achieved by FTICR-MS. Based on peak intensity, the relative abundance and the degree of
13
C labeling are calculated for individual
carotenoid isotopologs.
2 Materials
Prepare all aqueous solutions with ultrapure deionized water and
analytical grade (LC-MS grade) reagents. Discard remaining
extracts and solvents following waste disposal regulations.
264
Bjo ¨ rn Thiele and Shizue Matsubara