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Methodology combining mass spectrometry imaging (MSI) with ion mobility separation (IMS) has emerged as a biological imaging technique due to its versatility, sensitivity and label-free approach. This technique has been shown to separate isomeric compounds such as lipids, amino acids, carboxylic acids and carbohydrates. This report describes mass spectrometry imaging in combination with traveling-wave ion mobility separation and matrix-assisted laser desorption/ionization (MALDI). Positive ionization mode was used to locate fructans on tissue printed sections of  Agave  rhizome and stem tissue and distinguished fructan isoforms. Here we show the location of fructans ranging from DP3 to DP17 to be differentially abundant across the stem tissue and for the first time, experimental collision cross sections of endogenous fructan structures have been collected, revealing at least two isoforms for fructans of DP4, DP5, DP6, DP7, DP8, DP10, and DP11. This demonstrates that complex fructans such as agavins can be located and their isoforms resolved using a combination of MALDI, IMS, and MSI, without the need for extraction or derivatization. Use of this methodology uncovered patterns of fructan localization consistent with functional differences where higher DP fructans are found toward the central section of the stem supporting a role in long term carbohydrate storage whereas lower DP fructans are concentrated in the highly vascularized central core of rhizomes supporting a role in mobilization of carbohydrates from the mother plant to developing offsets. Tissue specific patterns of expression of genes encoding enzymes involved in fructan metabolism are consistent with fructan structures and localization.

Localization and Composition of Fructans in Stem and Rhizome of Agave tequilana Weber var. azul