Abstract
Cell walls of the wheat endosperm are mostly composed of arabinoxylans (AX) and mixed (1→3), (1→4)-β-glucans (BG) (Saulnier et al., 2012). Here, we present an optimized protocol to degrade enzymatically these cell-wall polysaccharides into oligosaccharides, directly from wheat grain cross sections. The main difficulty is to provide a sufficient amount of humidity for the enzyme to be active, while the amount of liquid at the surface of the tissue should stay low to prevent any delocalization of the released products. With this protocol, enzymatic degradation was shown to be efficient and delocalization of released oligosaccharides was estimated below 50 µm (Veličković et al., 2014). Although it can be employed for other purposes, this in situ enzymatic digestion was primarily developed to obtain molecular images of the cross-sections of wheat endosperm by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (Veličković et al., 2014). The cell wall polysaccharides are heterogeneous in structure, exhibit high masses and are entangled into complex networks. Thus, they are not amenable to direct analysis by mass spectrometry and they need to be degraded into smaller compounds as a first step. In this protocol, additional steps corresponding to the deposition of the MALDI matrix are also described.
Keywords: Enzyme application, Arabinoxylans, Beta glucans, MALDI MSI, Wheat section prepartaion
Materials and Reagents
Equipment
Procedure
Representative data
Recipes
Notes
Acknowledgments
This work was supported in part through a postdoctoral fellowship (Dušan Veličković) from INRA (Institut National de Recherche Agronomique, France) and AgreenSkills. We are very grateful to Fabienne Guillon and Luc Saulnier (INRA Biopolymers, Interaction, Assemblies, Nantes, France) for helpful discussions about the enzymatic hydrolysis of wheat cell walls. This protocol is a modified version of the protocol previously described in Veličković et al. (2014).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Hi Theodora, Thank you for your interest in our protocol! So, DMA/DHB matrix is by far the best MALDI matrix for carbohydrates (sugars and oligossacharides). You can find publication from our lab about this: http://onlinelibrary.wiley.com/doi/10.1002/rcm.5060/abstract There is theory that DMA mimics Schiff base formation with sugars which increase their sensitivity during ionization: http://onlinelibrary.wiley.com/doi/10.1002/rcm.3265/abstract For your second question, I usually scan sample before application of matrix. In this particular case I didn't stain it, but in fact you can stain it after finishing your MALDI run: wash matrix with 70% EtOH (by dipping plate in 70%EtOH solution 3x2min) and then you can stain it. Or you can stain consecutive section if this washing doesn't give you good results. For your stems: I did analyze long time ago xylans from maize stem: be aware that you need to know general chemical organization and polyssacharides composition. For example I did remember that these xylans in maize stem are very very rich in acetylation and feruliation so I needed to remove these decorations by KOH treatment before application of enzymes (since these decorations will hide glycosidic bond that enzyme recognizes). I hope this will guide your experiments. Feel free to contact me if you need further assistance. Dusan
Hi Theodora, Thanks for your questions! Filter paper is used to saturate air in chamber with solvent vapor, so that sample doesn't dry there. In fact, filter paper absorbs the liquid in the solvent and provides more surface area for evaporation. More surface area=faster evaporation. More evaporation means more solvent vapor in the chamber's air. A saturated solution of salt in water is used to maintain particular values of relative humidity inside chamber. K2SO4 gives the highest relative humidity. At 45C saturated K2SO4 has 96% relative humidity, which we used in our experiments. We tried also different salts solutions, but every time droplet of enzyme will not "survive" after overnight incubation. With K2SO4 we could incubate 24h without drying of enzyme droplet spotted on the tissue. I hope this will help you in your experiments, Dusan