A high-throughput matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry-based assay of...

A high-throughput matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) assay is described for determination of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates and is readily achievable on a microliter scale (2μl of total volume containing 2μg of substrate and 1ng of protein). The speed and sensitivity of the assay make it potentially well suited for the high-throughput screening of chitinase inhibitors. The mass spectrum is acquired in approximately 2min, as opposed to typically 30-40min for a single run with a high-performance liquid chromatography (HPLC)-based assay. By using the multiple-place MALDI MS targets, we estimate that 100 assays could be run in approximately 2-3h without needing to remove the target from the instrument. In addition, because the substrate and product chitomers are visualized simultaneously in the TOF spectrum, this gives immediate information about the cleavage site and mechanism of the enzyme under study. The assay was used to monitor the purification and transgenic expression of plant class IV chitinases. By performing the assay with chitomer substrates and C-glycoside chitomer analogs, the enzyme mechanism of the class IV chitinases is described for the first time.