In Vivo Chemical Analysis of Plant Sap from the Xylem and Single Parenchymal Cells by Capillary Microsampling Electrospray Ionization Mass Spectrometry

Abstract

In plants, long-distance transport of chemicals from source to sink takes place through the transfer of sap inside complex trafficking systems. Access to this information provides insight into the physiological responses that result from the interactions between the organism and its environment. In vivo analysis offers minimal perturbation to the physiology of the organism, thus providing information that represents the native physiological state more accurately. Here we describe capillary microsampling with electrospray ionization mass spectrometry (ESI-MS) for the in vivo analysis of xylem sap directly from plants. Initially, fast MS profiling was performed by ESI from the whole sap exuding from wounds of living plants in their native environment. This sap, however, originated from the xylem and phloem and included the cytosol of damaged cells. Combining capillary microsampling with ESI-MS enabled targeted sampling of the xylem sap and single parenchymal cells in the pith, thereby differentiating their chemical compositions. With this method we analyzed soybean plants infected by nitrogen-fixing bacteria and uninfected plants to investigate the effects of symbiosis on chemical transport through the sap. Infected plants exhibited higher abundances for certain nitrogen-containing metabolites in their sap, namely allantoin, allantoic acid, hydroxymethylglutamate, and methylene glutamate, compared to uninfected plants. Using capillary microsampling, we localized these compounds to the xylem, which indicated their transport from the roots to the upper parts of the plant. Differences between metabolite levels in sap from the infected and uninfected plants indicated that the transport of nitrogen-containing and other metabolites is regulated depending on the source of nitrogen supply.

Selective Capture-Based Single-Cell Mass Spectrometry for Enhancing Sphingolipid Profiling of Neurons with Differentiation of Cell Body from Synapse

“…10−13 However, they are generally operated under vacuum conditions and are not suitable for living cell analysis. In recent years, different ambient sampling methods coupled with electrospray ionization (ESI) were developed for single-cell analysis in ambient conditions or even in living cells, including directly drawing the cytoplasm from a cell using a micropipette, capillary microsampling 14 or patch clamp techniques, 15 ambient liquid extraction by droplet extraction, 16,17 singleprobe, 18,19 T-probe 20,21 and nanospray desorption ESI (nano-DESI), 22 and in vivo solid-phase microextraction (SPME) via probe ESI (PESI). 23,24 Among them, ambient liquid extraction techniques could achieve solvent extraction of cellular contents during sampling, resulting in higher coverage of insoluble compounds in or out of the cytoplasm.…”

“…Matrix-assisted laser desorption ionization (MALDI) and secondary ion MS (SIMS) are widely used spatially resolved ionization methods in single-cell analysis and have been used to provide phospholipid profiling of single cells and even the subcellular spatial distribution of phospholipids. − However, they are generally operated under vacuum conditions and are not suitable for living cell analysis. In recent years, different ambient sampling methods coupled with electrospray ionization (ESI) were developed for single-cell analysis in ambient conditions or even in living cells, including directly drawing the cytoplasm from a cell using a micropipette, capillary microsampling or patch clamp techniques, ambient liquid extraction by droplet extraction, , single-probe, , T-probe , and nanospray desorption ESI (nanoDESI), and in vivo solid-phase microextraction (SPME) via probe ESI (PESI). , Among them, ambient liquid extraction techniques could achieve solvent extraction of cellular contents during sampling, resulting in higher coverage of insoluble compounds in or out of the cytoplasm. Moreover, ambient liquid extraction techniques are flexible in solvent composition, making them advantageous for selective extraction, online capture, and standard addition quantitation .…”

Single‐Cell Metabolomics by Mass Spectrometry: Opportunities and Challenges

“…In the nanoPOTS technology, used mostly for single cell proteomics, cells isolated by fluorescence‐activated cell sorting are chemically processed in nanowells that provide a highly confined volume to avoid unnecessary dilution of the cell content [14] . Tissue embedded mammalian and plant cells can be interrogated by focused ion beams (secondary ion MS, SIMS, and multiplexed ion beam imaging, MIBI) or laser beams (matrix‐assisted laser desorption ionization, MALDI, and laser ablation electrospray ionization, LAESI), or capillary microsampling [15–17] …”

Hydrogen/Deuterium Exchange Aiding Metabolite Identification in Single-Cell Nanospray High-Resolution Mass Spectrometry Analysis

“…Mass spectrometry (MS) is an attractive technique for cellular and subcellular analyses of chemical species because of its high selectivity and sensitivity. Capillary microsampling electrospray ionization mass spectrometry (ESI-MS) and other ambient single-cell MS technologies have been developed − to directly obtain molecular information about biological cells with little sample preparation. Nowadays, capillary microsampling combined with electrophoresis (CE)-MS is capable of metabolite analysis at the single-cell level .…”