Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Summary

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Abstract

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Introduction

Plants contain a wide range of small molecules with diverse chemical properties. MS is a powerful technique for analyzing plant compounds because it can provide elemental compositions with a high sensitivity and specificity for the detection and identification of metabolites¹. Most commonly, MS is performed on solvent-extracted samples, which are separated by chromatography prior to the MS analysis¹. However, the use of liquid chromatography (LC) requires lengthy analysis times and is often associated with an extensive sample preparation¹. In contrast, direct chemical analysis of intact tissues th....

Protocol

1. Modifications to Nanospray Source for Leaf Spray MS

1. Use a modified nanospray source for leaf spray MS. As no fluidic components are necessary for leaf spray MS, modify the source by removing the LC probe from the source.
2. Assemble the leaf spray MS wire that will apply the voltage to the plant tissue with the appropriate pin for plugging into the source. Solder the pin to one end of an insulated wire; solder a clamp to the opposite end of the wire.
   NOTE: The clamp (alligator clip type) may or may not have teeth. For small tissue, a clamp without teeth is preferred. An optional flex arm with a clamp may be added to the nanospray source to....

Results

At 10 weeks post-germination, freshly collected greenhouse-grown S. tortuosum leaves were analyzed by leaf spray MS. The experimental workflow for detecting metabolites from S. tortuosum leaves using leaf spray MS is illustrated in Figure 2. A leaf was selected, cut into a thin strip with a tapered end to form a point, and clamped with the leaf spray MS wire clamp apparatus. The plant tissue was positioned ~30 mm from the ion inlet and in li.......

Discussion

The successful use of this protocol relies on the optimization of various steps for the plant species, tissue type, and target compound(s) of interest. The parameters described in the protocol provide a good starting point. The following experimental decisions need to be made and tested: whether or not to use (1) cut or uncut tissue and (2) solvent or no solvent, (3) what solvent to use and in what volume, (4) what the distance of tissue from the ion inlet should be, and (5) the voltage amplitude. The goal of optimizatio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Conn Pin	Digi-Key elctronics	WM2563CT-ND	pin will insert into Thermo Scientific source to provide voltage
small clamp	Digi-Key elctronics	314-1018-ND	CLIP MICRO ALLIGATOR COPPER 5A
large clamp	Digi-Key elctronics	290-1951-ND	ALLIGATOR CLIP NARROW NICKLE 5A
Heat shrink	Digi-Key elctronics	Q2Z1-KIT-ND	to cover soldering joints
NSI source Nanospray Ion Source	Thermo scientific	NA	Another brand will work if you are not using a Thermo instrument
Q Exactive- hybrid quadrupole Orbitrap	Thermo scientific	NA	Another brand will work if you are not using a Thermo instrument
Tune Software	Thermo scientific		Another brand will work if you are not using a Thermo instrument
Xcalibur Software	Thermo scientific
Plant of interest - S. tortousum