Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/20/2010
Publication Date: 12/25/2010
Publication URL: http://hdl.handle.net/10113/48557
Citation: Price, N.P., Naumann, T.A. 2011. A high-throughput MALDI-TOF mass spectrometry-based assay of chitinase activity. Analytical Biochemistry. 411(1):94-99.
Interpretive Summary: Plant chitinases break down the cell walls of fungal and insect pathogens, and are important targets for the design of new anti-fungal agents and insecticides. This paper describes a new assay to measure the activity of plant chitinases. The assay uses mass spectrometry to measure the differences in the mass of degraded chitin verses non-degraded. It can be used to compare different chitinases, and to measure chitinase rates over a period of time. The new assay has been used to purify several chitinases from corn (maize) which are involved in resistance to corn pathogens. The assay described in this work will be valuable to researchers involved in the development of anti-fungal agents.
Technical Abstract: A high-throughput MALDI-TOF mass spectrometric assay is described for assay of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates, and is readily achievable on a microliter scale (2 µL total volume, containing 2 µg of substrate and 1 ng of protein). The speed and sensitivity of the assay make it well suited for the high-throughput screening of chitinase inhibitors. The mass spectrum is acquired in approximately two minutes, as opposed to typically 30-40 minutes for a single run for an HPLC-based assay. By using the multiple-place MALDI MS targets we estimate that one hundred assays could be run in about 2-3 hours, without needing to remove the target from the instrument. In addition, because the substrate and product chitomers are simultaneously visualized in the time-of-flight spectrum this gives immediate information about the cleavage site and mechanism of the enzyme under study. The assay has been 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.