Identification and Localization of Labile Molecules in Broccoli Cell by CLA-ICP-MS and Single-Cell Nanospray HRMS.

Although molecular analysis and imaging by mass spectrometry are emerging as tools to identify metabolites and determine their distribution in cells and tissues, it is difficult to directly analyze the labile molecules at the single-cell level. Glucosinolate (GL) is a plant-active substance with much attention as a chemical defense mechanism known as a "mustard oil bomb" in broccoli. When tissue is damaged, these substances undergo rapid degradation, making them unsuitable for conventional mass spectrometry (MS), particularly for surface MS imaging analysis methods that necessitate intricate preprocessing. Herein, a strategy combining cryogenic laser ablation inductively coupled mass spectrometry (CLA-ICP-MS) and capillary microsampling nanospray high-resolution mass spectrometry (HRMS) was developed. The sulfur-rich microzone in tissue which was thought as a suspect GL-rich cell population was located via CLA-ICP-MS. Three GLs in single cells were accurately identified by nanospray HRMS with a hydrogen/deuterium exchange reaction. Subsequently, cell-by-cell imaging by nanospray HRMS showed that the GL-rich cells were below the stalk surface by approximately 30 µm. This proposed strategy can also be applied to rapidly identify labile compounds and localize molecule-rich cells in tissues.

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

The identification of metabolites in single-cell or small-volume tissue samples using single-cell mass spectrometry (MS) is challenging. In this study, hydrogen/deuterium (H/D) exchange was combined with microsampling nanospray high-resolution mass spectrometry (HRMS) to improve the efficiency and confidence level of metabolite identification in a single cell using commercial software. A nanospray ion source showed an improved reaction depth of 8% for H/D exchange compared with an electrospray ion source. In total, 273 metabolites were identified in Allium cepa L. single cells by searching commercial databases. Generally, more than one candidate is given for a precursor ion by MS or tandem MS (MS2) databases such as ChemSpider, MetDNA, MassBank, and mzCloud. With the help of the H/D exchange technique, the number of candidates decreased and reduction of the search space by a factor of 8 was achieved. In addition, two enzymolysis products of isoalliin, the transient intermediate and its isomer, were tracked at the single-cell level using the proposed method.