Combined use of cutinase and high-resolution mass-spectrometry to query the molecular architecture of cutin.

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

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.