High-throughput functional assessment of polysaccharide-active enzymes using matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry as exemplified on plant cell wall polysaccharides

Despite a wealth of sequence information on genes encoding carbohydrate-active enzymes (e.g., transferases, esterases, hydrolases), very few of these enzymes have been described in detail, particularly regarding substrate specificities. A facile and rapid method for the characterization of substrate specificities of polysaccharide-active enzymes that uses matrix-assisted laser desorption–time of flight mass spectrometry (MALDI–TOF MS) has been developed. This method has been applied to characterize a xyloglucan fucosyltransferase and a pectin methyl-esterase. Reactions were performed in liquid phase, and aliquots of the reaction mixtures were spotted on a polyvinylidene fluoride (PVDF) membrane. Reaction products were precipitated onto the membrane and cleaned by treatment with an ethanol–water mixture. Subsequently, the reaction products were hydrolyzed by specific endoglycanases, and the resulting oligosaccharides were directly analyzed onto the PVDF membrane by MALDI–TOF MS. The new method is amenable to high-throughput analysis and, thus, constitutes an emerging avenue to rapidly fill the gap in our knowledge of the specificities of polysaccharide-active enzymes.