In situ Digestion of Wheat Cell-wall Polysaccharides

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.