Combining DLEMMA with MALDI MSI for Spatial Analysis of Plant Metabolites

Liron Feldberg, Department of Plant Sciences, The Weizmann Institute, Rehovot, Israel
Dong Yonghui, Department Of Plant Sciences, The Weizmann Institute, Rehovot, Israel
Uwe Heinig, Department of Plant Sciences, The Weizmann Institute, Rehovot, Israel
Ilana Rogachev, Department of Plant Sciences, The Weizmann Institute, Rehovot, Israel
Asaph Aharoni, Department Of Plant Sciences, The Weizmann Institute, Rehovot, Israel

Dual Labeling of Metabolites for Metabolome Analysis (DLEMMA) coupled with LC-MS, previously developed in our lab, is an indispensable metabolomics tool for metabolites identification and network construction. It enables tracking a particular metabolic pathway by enriching the concentrations of metabolites in this pathway with precursor feeding, and helping in structure elucidation according to their labeling patterns obtained from MS spectra. Mass spectrometry imaging (MSI) is a molecular imaging techniques, which allows direct localization of a large variety of molecules from tissue sample surfaces without any sample extraction. The significance of being able to map the distribution of molecules in a multiplex manner and with limited a priori knowledge has allowed it an attractive molecular histology tool.

Coupling DLEMMA with MSI provides the following advantages: 1) Increased metabolites concentrations derived from the fed precursor. Since MSI technique luck chromatographic separation, signal suppression might be high. Hence, increasing metabolite concentration is significant for MSI detection. 2) Improved confidence in metabolite identification and spatial localization based on localization pattern of the unlabeled and labeled metabolites in MSI analysis. In MSI analysis, Isomers (same m/z with different chemical structure) can't be distinguished and false identification and localization might occurred when more than one abundant isomer exist in the sample, therefore, similar localization pattern of unlabeled and labeled metabolites provide additional parameter for metabolite identification and ensure reliable spatial localization.

Combining the information obtained from coupling DLEMMA with both analysis method, MSI and LC-MS, increase metabolites coverage derived from the fed precursor, enhance the confidence in metabolites detection, identification and localization (according to exact m/z, labeling pattern, clustering masses with same  R.T to find the parent ion, localization pattern and ms-ms data) and provide spatial distribution of metabolites in intact tissue, which is valuable to improve our understanding of biological processes.  

The application of DLEMMA coupled to MALDI-MSI and LC-MS was demonstrated with two plant tissues, Lemna (i.e. Spirodela polyrhiza) and tomato fruit. They were fed with unlabeled and two different labeled stable isotopes (Lemna with unlabeled tyrosine, tyrosine D4 and tyrosine 13C915N and, tomato fruit with unlabeled phenylalanine, phenylalanine D5 and phenylalanine 13C915N). More than 40 amino acid derived metabolites were identified in each tissue and their localization was visualized by MSI.

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