Abstract
BackgroundCutin is a complex, highly cross-linked polyester consisting of hydroxylated and epoxidated acyl lipid monomers. Because of the complexity of the polymer it has been difficult to define the chemical architecture of the polymer, which has further limited the ability to identify the catalytic components that assemble the polymer. Analogous to methods that define the structure of oligosaccharides, we demonstrate a strategy that utilizes cutinase to generate cutin subfragments consisting of up to four monomeric units, whose structure and spatial distribution in the polymer is revealed by high-resolution mass spectrometry. Moreover, the application of mass-spectrometric fragmentation and labelling of the end of the oligomers, one is able to define the order of monomers in the oligomer. The systematic application of this strategy can greatly facilitate understanding the chemical architecture of this complex polymer.ResultsThe chemical architecture of plant cutin is dissected by coupling an enzymatic system that deconstructs the polymer into subfragments consisting of dimers, trimers and tetramers of cutin monomers, with group-specific labeling and mass spectrometry. These subfragments can be generated with one of over 1200 of cutinases identified from diverse biological sources. The parallel chemical labeling of the polymer with dansyl, alkyl or p-dimethylaminophenacyl reagents can identify the chemical distribution of non-esterified hydroxyl- and carboxyl-groups among the monomers. This combined strategy is applied to cutin isolated from with apple fruit skins, and a combination of gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–quadrupole time-of-flight (Q-TOF) MS is used to determine the order of the monomers in the cutinase-generated subfragments. Finally, we demonstrate the use of matrix-assisted laser desorption-ionization-MS to determine the spatial distribution of the cutinase-generated subfragments.ConclusionOur experimental results demonstrate an advancement to overcome the current limitations in identifying cutin oligomeric structure and allows one to more efficiently address new biological questions about cutin biosynthesis. We submit that the systematic application of these methods will enable the construction of more accurate architectural models of cutin, which is a prerequisite to identifying cutin-biosynthetic components.
Highlights
Cutin is a complex, highly cross-linked polyester consisting of hydroxylated and epoxidated acyl lipid monomers
The purified cutinases were determined to be greater than 95% homogeneous by SDS-PAGE (Additional file 2: Fig. S1), and they migrated within the expected molecular weight range of between 24 and 30 kDa
We applied methods that can systematically characterize the bonding connections and patterns of the monomeric units within cutin by the combined use of cutin (See figure on page.) Fig. 5 a Relative ratio of dansylated and non-dansylated cutin-monomers recovered from cutinase hydrolysis of dansylated intact cutin. b Recovery of alkylated and nonalkylated cutin-monomers generated by the hydrolysis of benzyl-O-alkylated intact cutin. c Relative ratio of DmPA-derivatized and non-derivatized cutin-monomers recovered from hydrolysis of DmPA-treated intact cutin
Summary
Highly cross-linked polyester consisting of hydroxylated and epoxidated acyl lipid monomers. Because of the complexity of the polymer it has been difficult to define the chemical architecture of the polymer, which has further limited the ability to identify the catalytic components that assemble the polymer. Despite extensive knowledge of the monomeric constituents of cutin [5, 6], the complexities of its polymeric structure and its inter-connections with other cellular components has hindered the ability to understand how cutin is assembled and how it serves its biological function [7, 8]. Advancements have been made in identifying the architecture of cutin using solid state NMR and mass spectrometric analysis of soluble cutin oligomers following acid- or base-catalyzed depolymerization of cutin [9, 10], a comprehensive higher order structure of this important polymer is still unknown
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