HR-μMAS NMR-Based Metabolomics: Localized Metabolic Profiling of a Garlic Clove with μg Tissues

Abstract

The localization of metabolic profiles within a tissue sample is of particular interest when the sampling size is considerably small, i.e. in the order of microgram (µg) scale. Small sampling size is inevitable when the sample availability is limited, or when different metabolic profiles are suspected in small disparate sample regions. Capitalizing a recently introduced high-resolution micro-MAS probe (HR-µMAS) for its capability of high-quality NMR data acquisition of µg samples, this study explores the localized metabolic NMR profiling of a single garlic clove and compares the methodology and results with the standard HR-MAS. One advantage of HR-µMAS is the feasibility of analyzing homogenous µg samples within a large heterogeneous tissue. As a result, the sampling mass (< 0.5 mg) allows to selectively profile four homogenous anatomic garlic regions by HR-µMAS (skin, flesh, inner epidermis and sprout), in contrast to three regions (skin, flesh and core ≡ inner epidermis & sprout) by HR-MAS with a sampling mass of ca. 8 mg. Discriminant analysis was carried out to identify the significant variables in the different regions. It found a significant presence of fructose in both skin and flesh, while sucrose and glucose are predominant in the core. Among the garlic characteristic sulfur compounds, allicin is dominant in the core and allyl mercaptan in both skin and flesh. Quantification analysis was conducted and demonstrated its potential by quantifying metabolites at µg-level. This study offers an important basis for designing HR-µMAS NMR-based metabolomics studies that can benefit from profiling µg-scale samples.

NMR Spectroscopy for Metabolomics Research

“…In particular, microprobes for magic-angle spinning (μMAS) of sub-microgram specimens with high-resolution (<0.01 ppm) capability have recently appeared [192]. These permit the site-specific metabolomic characterization of different anatomical regions of plants and tissues [193]. While sample preparation can be difficult, the capabilities of μMAS probes gives NMR spectroscopists the opportunity to systematically explore tissue-specific metabolism with nearly equal precision as imaging mass spectrometry [194].…”

NMR-based metabolomics and fluxomics: developments and future prospects

“…23 Microprobes have also been developed under HR-MAS (high-resolution magic angle spinning) configurations, in order to work on small amounts of tissue samples. 80 Still with the aim to pursue the quest for a sensitive NMR detection of metabolites in masslimited samples, recent developments in the field of microfluidics seem particularly promising.…”

Current Developments in µMAS NMR Analysis for Metabolomics

“…Capitalizing the merits of HR-µMAS, HR-µMAS NMR was successfully applied to comprehensive metabolic profiling of a food tissue [30]. Figure 5a shows the sampling of µg tissues with good spectral quality data (15-min acquisition with 0.03 ppm resolution), permitting localized profiling of the different anatomical regions of a garlic clove: skin, flesh, core-sprout and core-inner epidermis.…”

“…With OPLS-DA quality-parameters: 1 predictive component and 1 orthogonal component, R 2 X = 0.747, R 2 Y = 0.955, Q 2 = 0.828, and a p-value of the cross-validation = 0.027. Adapted with permission from [30]. Copyright (2018) American Chemical Society.…”