Electrospray/Photochemical Ionisation Mass Spectrometry Imaging Source (DESI/PI) for in situ imaging of metabolites in plant leaves

Areas of application: plant metabolomics; traditional Chinese medicine; food processing;

Introduction

Plant leaves contain secondary metabolites with widely varying polarities, such as terpenoids, flavonoids, glycosides, phenolics and lipids. Their spatial distribution is closely related to biosynthetic pathways and physiological regulatory mechanisms, making them a key focus of research in the food and pharmaceutical sectors. Conventional electrospray ionisation mass spectrometry imaging (DESI-MSI) is affected by the plant epidermal wax barrier, ionisation polarity limitations and ion suppression effects; consequently, the ionisation efficiency of weakly polar metabolites is extremely low, making it difficult to detect a large number of key bioactive compounds.

Physical dewaxing pretreatment disrupts the original spatial distribution of metabolites, making true in situ imaging unfeasible. The imprinting method, combined with electrospray/photochemical ionisation (DESI/PI) mass spectrometry imaging, integrates pressure-transfer sample preparation with vacuum ultraviolet post-ionisation technology, fully preserves the spatial information of metabolites under ambient pressure. Through photon-induced secondary ionisation, the detection range and signal intensity of moderately polar and weakly polar metabolites are significantly enhanced, enabling in situ imaging of hundreds of metabolites in fresh plant leaves and providing a novel characterisation method for elucidating plant metabolic synthesis sites and transformation pathways.

Materials and Methods

Instrumentation: electrospray/photochemical ionisation mass spectrometry imaging source (DESI/PI), Agilent 6224 mass spectrometer (resolution 20,000).

Reagents: chromatographically pure methanol, toluene, formic acid, nitrogen gas, porous PTFE membranes.

Plant samples: fresh sage leaves, ginkgo leaves, fresh green tea leaves.

‌Results and Conclusions

1. Post-ionisation (PI) significantly enhances metabolite detection coverage and signal sensitivity

DESI alone can only efficiently detect highly polar compounds; the signals of a large number of weakly polar secondary metabolites, such as terpenes, alkylphenols and flavonoids, are suppressed; Upon the introduction of post-ionisation (PI), the neutral molecules generated by DESI desorption and ionisation undergo secondary ionisation via photon-induced ion–molecule reactions, resulting in simultaneous performance improvements in both positive and negative ion modes.

Positive ion mode: Compared with conventional DESI, DESI/PI detected over 80 additional metabolites; imaging signals for characteristic bioactive compounds such as terpenes, ginkgolides, catechins and flavonoid glycosides were newly observed in samples of sage leaves, ginkgo leaves and tea leaves.

Negative ion mode: The signal intensity of most metabolites increased by 1–2 orders of magnitude, with the signals for some substances amplified by 20–40 times. Only highly polar lipids such as choline and phosphatidylcholine experienced slight signal suppression due to the weakly polar toluene in the spray solvent; for all other metabolites, the signal-to-noise ratio in the imaging was significantly superior to that of single DESI.

Figure 1 Schematic diagram of the experimental workflow for plant leaf imprint mass spectrometry imaging

2. Spatial distribution imaging of metabolites in typical plant leaves

(1) Sage leaves

Diterpenes and triterpenes with antioxidant properties—such as dehydrocurcumic acid, curcumic acid, salicylic acid, ursolic acid and oleanolic acid—are evenly distributed throughout the mesophyll, whilst their concentrations in the vein regions are extremely low; glycerol diester neutral lipids are primarily concentrated in the veins. Figure 2(C) provides a clear visual representation of the regions where anti-inflammatory and anti-cancer active compounds of the terpenoid class are synthesised.

Figure 2 (A) Average mass spectra of sage leaves imprinted using DESI/PI (red) and DESI (black). (B) Vewin plots of compounds detected via imprinted DESI/PI and DESI in positive ion mode. (C) MS images of some representative compounds in sage leaves.

(2) Ginkgo leaves

Flavonoids (shannaiol, luteolin, quercetin) are uniformly distributed throughout the mesophyll tissue; alkylphenols such as ginkgol and ginkgolic acid are specifically localised in the secretory cavities of the leaves, with their spatial distributions being complementary; Figure 3(C) clearly distinguishes the sites of synthesis for the pharmacologically active flavonoids in Ginkgo from those for the alkylphenols that cause toxic side effects, thereby providing a basis for quality control of Ginkgo medicinal materials.

Figure 3 (A) Average mass spectra of Ginkgo leaves imprinted using DESI/PI (red) and DESI (black). (B) Venn diagram of compounds detected via imprinted DESI/PI and DESI in positive ion mode. (C) MS images of some representative compounds in ginkgo leaves.

(3) Fresh green tea leaves

Caffeine is concentrated in the midrib and diffuses into the leaf mesophyll, confirming that caffeine in tea leaves is primarily synthesised in the tender tissues of the veins; theanine is heavily concentrated in the petiole and midrib, confirming that theanine is transported from the root system to the leaves via the vascular system.

The abundance of various catechins (EC/EGC/ECG/EGCG) in the leaf vein region is 2 to 6 times that of the leaf mesophyll; key intermediate products such as gallic acid, β-glucose and gallic acid are also concentrated in the leaf veins, directly proving that the central region of green tea catechin biosynthesis is the main vein of the leaf.

Figure 4 (A) MS images of some representative metabolites in tea leaves detected via DESI/PI imprinting in positive ion mode. (B) Average mass spectra of tea leaves obtained via DESI/PI (red) and DESI (black). (C) MS images of some representative metabolites in tea leaves detected via DESI imprinting.

Conclusions

The combination of imprinting with electrospray/photochemical ionisation (DESI/PI) mass spectrometry imaging circumvents interference from the waxy barrier on plant cuticles, allowing the complete preservation of the original spatial information of metabolites without the need for chemical dewaxing; the integration of DESI with photon secondary ionisation enables compatibility with metabolites across the entire polarity range, overcoming the polarity discrimination limitations of conventional DESI ionisation.

This method has successfully achieved simultaneous imaging of hundreds of metabolites in the leaves of three typical plant species—sage, ginkgo and tea—enabling the intuitive analysis of secondary metabolite synthesis sites, transport patterns and biosynthetic networks. It provides a highly sensitive in situ visualisation tool for research into plant stress response physiology, bioactive compounds in medicinal plants and metabolic pathways of plant natural products.

References

[1] Liutian Wu, Keke Qi, Chengyuan Liu*, Yonghua Hu, Minggao Xu, Yang Pan*, Enhanced coverage and sensitivity of imprint DESI Mass Spectrometry Imaging for Plant Leaf Metabolites by Post-Photoionisation, Analytical Chemistry, 2022, 94, 15108–15116