Chapter 20
Carotenoid Isotopolog Profiling in
13 C-Labeled Leaf Extracts
by LC-MS and LC-FTICR-MS
Bjo ¨ rn Thiele and Shizue Matsubara
Abstract
Mass spectrometry (MS)-based metabolite analysis combined with stable isotope labeling offers a powerful
tool to study dynamic regulation of metabolic pathways and metabolite fluxes in biological systems. Here
we describe a method to analyze the composition of carotenoid isotopologs in
13 C-labeled leaf extracts by
using liquid chromatography (LC)-MS and LC-Fourier transform ion cyclotron resonance (FTICR)-MS.
High mass resolution of the latter enables unambiguous assignment of observed mass to a unique chemical
formula. Based on peak intensity the relative abundance and the degree of
13 C labeling are calculated for
individual carotenoid isotopologs.
Key words
13 C labeling, Carotenoid metabolism, Degree of labeling, FTICR-MS, Isotopolog
profiling, LC-MS
1 Introduction
Plants are a rich source of naturally occurring carotenoids. They
accumulate the yellow to orange-reddish pigments in fruits and
flowers to attract pollinators and seed dispersers. In leaves carotenoids play a dual role, collecting sunlight in the blue wavelength
region to drive photosynthesis, while at the same time protecting
the photosynthetic membrane against photooxidation. Furthermore, some carotenoids can serve as precursors to produce plant
hormones, signaling agents, and fragrances. Owing to these important functions in plants, biosynthesis and degradation of carotenoids have been studied intensively by researchers [1, 2], aided by
analytical tools to identify and quantify pathway products and
intermediates.
Stable isotope labeling was a key technique in the early days of
biochemical pathway analysis, including the biosynthesis of isoprenoids and carotenoids [3, 4]. Besides using specifically labeled
organic tracers, carbon fluxes in plants can be studied in vivo by
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_20,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
263
Carotenoid Isotopolog Profiling in
13 C-Labeled Leaf Extracts
by LC-MS and LC-FTICR-MS
Bjo ¨ rn Thiele and Shizue Matsubara
Abstract
Mass spectrometry (MS)-based metabolite analysis combined with stable isotope labeling offers a powerful
tool to study dynamic regulation of metabolic pathways and metabolite fluxes in biological systems. Here
we describe a method to analyze the composition of carotenoid isotopologs in
13 C-labeled leaf extracts by
using liquid chromatography (LC)-MS and LC-Fourier transform ion cyclotron resonance (FTICR)-MS.
High mass resolution of the latter enables unambiguous assignment of observed mass to a unique chemical
formula. Based on peak intensity the relative abundance and the degree of
13 C labeling are calculated for
individual carotenoid isotopologs.
Key words
13 C labeling, Carotenoid metabolism, Degree of labeling, FTICR-MS, Isotopolog
profiling, LC-MS
1 Introduction
Plants are a rich source of naturally occurring carotenoids. They
accumulate the yellow to orange-reddish pigments in fruits and
flowers to attract pollinators and seed dispersers. In leaves carotenoids play a dual role, collecting sunlight in the blue wavelength
region to drive photosynthesis, while at the same time protecting
the photosynthetic membrane against photooxidation. Furthermore, some carotenoids can serve as precursors to produce plant
hormones, signaling agents, and fragrances. Owing to these important functions in plants, biosynthesis and degradation of carotenoids have been studied intensively by researchers [1, 2], aided by
analytical tools to identify and quantify pathway products and
intermediates.
Stable isotope labeling was a key technique in the early days of
biochemical pathway analysis, including the biosynthesis of isoprenoids and carotenoids [3, 4]. Besides using specifically labeled
organic tracers, carbon fluxes in plants can be studied in vivo by
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_20,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
263