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

Leaf spray MS is a fast method for qualitative and semi-quantitative analysis of intact plant tissues that has a wide variety of applications. The main advantage of this technique is plant tissue can be analyzed directly with essentially no sample preparation. Although this method provides good insights into mesembrine alkaloids from Sceletium, it can also be used for analysis of various other plant species and exogenous chemicals such as pesticides.

Individuals new to this method may find it challenging because it is necessary to optimize the protocol for each plant species, tissue type, and compound of interest. Use a modified nano spray source for leaf spray MS.As no fluidic components are necessary for leaf spray MS, modify the source by removing the liquid chromatography probe from the source. The leaf spray mass spectrometry wire that will apply voltage to the plant tissue has a pin that has been soldered to the wire on one end and a clamp that has been soldered on the other end.

If the system has recently been in use, allow it to cool to the touch before removing any alternative source in the sweep cone. Then attach the nano spray leaf spray MS source. Finally, create a tune file and a method file as described in the text protocol.

Always wear gloves and do not use plant tissue that has been handled with bare hands. Otherwise, contaminant ions such as polyethylene glycol will dominate the spectra. Bring the plant tissues for the analysis to the same room as the mass spectrometry system to allow for a rapid sampling.

For plant tissue that does not have a naturally pointed tip, use a razor blade on a glass slide to cut a tapered point. Cut Sceletium tortuosum leaves at 10 weeks post germination into thin strips each with a tapered end to form a point. Use forceps to gently select the plant tissue at the end that will be clamped.

Holding the tissue with forceps, carefully transfer it to the clamp. Adjust the flexible arm and the wire with the clamp to position the tissue in line with the MS inlet. Refer to the text for the optimal distance from the ion inlet for each specific mass spectrometer.

If the first attempts produce a low signal intensity, the plant tissue can be moved closer to the ion inlet. Load the method file. Name the data file and set the file storage location.

Then turn on the MS system by clicking play and click on start to begin acquiring data. Apply a solvent using a pipette with a gel loading tip to maximize the distance between the high voltage and the user's hands for protection. Acquire data as long as the signal persists or until the adequate spectra have been collected, typically 30 to 60 seconds.

Stop the data acquisition and pause the MS system. Following acquisition, remove the tissue and wash the clamp with 100%methanol in a lint-free wipe. Open the data file and visually inspect the base peak mass chronogram.

Check that the signal intensity is approximately 10 million to 500 million. Make a new method file with an inclusion list of M over Z out to four decimal places. Click on global lists and inclusion.

Under properties of PRM, select the fragmentation energy and other tandem MS parameters. To obtain the tandem MS data for mesembrine alkaloids, fragment the ions at 276.1583 M over Z, 290.1742 M over Z, and 292.1897 M over Z at a normalized collision energy of 35. Next, load the tandem MS method file and a named data file.

Turn on the MS system and start acquiring data adding a solvent if necessary. When adequate spectra have been collected, typically after 30 to 60 seconds, stop the acquisition. Perform data analysis as described in the text protocol.

Representative results of metabolic profiling of Sceletium tortuosum by leaf spray MS and positive ionization are shown with the inset displaying 260 to 295 M over Z.In this study, a total of nine mesembrine alkaloids were detected by leaf spray MS and puditive identifications were made. The leaf spray MS total ion count mass chronogram represents a successful leaf spray MS experiment where a stable signal was maintained for the full duration of the acquisition. For each peak, the top number is the time in minutes and the bottom is the M over Z.Tandem mass spectra were collected in positive ionization made from a Sceletium tortuosum cut leaf with leaf spray MS.Puditive identifications of the alkaloids were made from accurate mass and mass fragmentation for mesembrine M and mesembrenone M isomers, mesembrine and mesembrine M dihydro.

After watching this video, you should have a good understanding of how to use leaf spray MS to detect plant metabolites. Leaf spray MS has been used to detect endogenous compounds as well as exogenous compounds such as pesticides.