Background

In many flowering plants xyloglucan is a major component of primary cell walls, where it plays an important role in growth regulation. Sequential extraction protocols offer a way of separating distinct xyloglucan domains (Pauly et al., 1999). Some xyloglucan appears to be trapped inside cellulose microfibrils while another fraction is bound to their surface through hydrogen bonding. The rest of the xyloglucan, possibly the majority of it, occupies the space between microfibrils (Park and Cosgrove, 2015). Part of this later xyloglucan can be extracted through direct endoglucanase digestion of cell wall material. Much of the remaining xyloglucan can then be released through alkaline treatment. Studies of mutants deficient in xyloglucan exoglycosidases have shown that these enzymes, together with Xyloglucan Endotransglycosylases/Hydrolases, act mostly on the enzyme-accessible fraction (Sampedro et al., 2010; Günl and Pauly, 2011; Günl et al., 2011; Sampedro et al., 2012; Sampedro et al., 2017). Digestion of Arabidopsis xyloglucan with endoglucanases that attack unsubstituted glucose residues results in a mixture of three and four-glucose subunits that can be quickly and easily analyzed through MALDI-TOF mass spectrometry (Lerouxel et al., 2002; Günl et al., 2010). The area of the ion peaks can be then used to quantify the abundance of the different fragment, although there is evidence of significant differences in response factors (Tuomivaara et al., 2015). This protocol incorporates some changes from our previous versions, such as the use of SDHB (Super-DHB) matrix, which reduces the noise, and the addition of NaCl during extraction to prevent the formation of potassium adducts.