Mass spectrometry based imaging of labile glucosides in plants

Mass spectrometry based imaging is a powerful tool to investigate the spatial distribution of a broad range of metabolites across a variety of sample types. The recent developments in instrumentation and computing capabilities have increased the mass range, sensitivity and resolution and rendered sample preparation the limiting step for further improvements. Sample preparation involves sectioning and mounting followed by selection and application of matrix. In plant tissues, labile small molecules and specialized metabolites are subject to degradation upon mechanical disruption of plant tissues. In this study, the benefits of cryo-sectioning, stabilization of fragile tissues and optimal application of the matrix to improve the results from MALDI mass spectrometry imaging (MSI) is investigated with hydroxynitrile glucosides as the main experimental system. Denatured albumin proved an excellent agent for stabilizing fragile tissues such as Lotus japonicus leaves. In stem cross sections of Manihot esculenta, maintaining the samples frozen throughout the sectioning process and preparation of the samples by freeze drying enhanced the obtained signal intensity by twofold to fourfold. Deposition of the matrix by sublimation improved the spatial information obtained compared to spray. The imaging demonstrated that the cyanogenic glucosides (CNglcs) were localized in the vascular tissues in old stems of M. esculenta and in the periderm and vascular tissues of tubers. In MALDI mass spectrometry, the imaged compounds are solely identified by their m/z ratio. L. japonicus MG20 and the mutant cyd1 that is devoid of hydroxynitrile glucosides were used as negative controls to verify the assignment of the observed masses to linamarin, lotaustralin, and linamarin acid