TITLE: Leaf surface acylsugar extraction and LC-MS profiling - v1.0
AUTHORS: Yann-Ru Lou, Bryan Leong

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">While this method is optimized for acylsugar extraction, it can be tweaked to use for other metabolites on the leaf surface.</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">Below is a literature that is highly useful for acyslugar identification:</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Comparative structural profiling of trichome specialized metabolites in tomato (</span><span style = "font-style:italic;">Solanum</span><span style = "font-style:italic;font-style:italic;"> </span><span style = "font-style:italic;">lycopersicum</span><span>) and </span><span style = "font-style:italic;">S. habrochaites</span><span>: acylsugar profiles revealed by UHPLC/MS and NMR (doi: https://doi.org/10.1007/s11306-013-0585-y)</span></li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Below is a literature that is highly useful for acyslugar identification:</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Comparative structural profiling of trichome specialized metabolites in tomato (</span><span style = "font-style:italic;">Solanum</span><span style = "font-style:italic;font-style:italic;"> </span><span style = "font-style:italic;">lycopersicum</span><span>) and </span><span style = "font-style:italic;">S. habrochaites</span><span>: acylsugar profiles revealed by UHPLC/MS and NMR (doi: https://doi.org/10.1007/s11306-013-0585-y)</span></li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div></div>

[STEPS]
?. [Reagent preparation]
Prepare the Acylsugar Extraction Solution (3:3:2 Acetonitrile:Isopropanol:water, 0.1% formic acid, 1 µM telmisartan (internal standard))To prepare Acylsugar Extraction Solution, combine:AcetonitrileIsopropanolddH2OFormic Acid (88%) (0.1% final conc.)1 mM Telmisartan (dissolved in 100% DMSO; final conc. 1 μM)
1000 ml
375 ml
375 ml
250 ml
1136 µl
1000 µl
We suggest always making fresh solution. However, in our experience, extraction buffer could be stored short term at 4℃ or -20℃ with no obvious issues. We did not carry out degradation curve and do not claim to know the best storage condition.
?. [Reagent preparation]
Dispense 1 ml of Acylsugar Extraction Solution into the labeled extraction vial
?. [Acylsugar extraction]
Using a fine scissors or forceps, cut 1-2 small new leaflets and immediately place them in the designated extraction vial
Clean the forceps and/or scissors with Acetonitrile and a Kimwipe between samples.
?. [Acylsugar extraction]
Tighten the cap well and gently agitate the tube on a rocker for
?. [Acylsugar extraction]
Remove the vial from the rocker and immediately pipette the extraction solution into the corresponding LC-MS glass vial and seal it with the cap.
Make sure the leaflet remains in the extraction vial.Acylsugars are not green, however, disturbance of chlorophyll and/or other metabolites might make the extraction solution turn green-ish looking. Based on our experience, this does not affect the extraction and qualification of acylsugars.
?. [Acylsugar extraction]
Store the LC-MS vials at -20 °C.For quantification purpose:Place uncapped extraction vials (containing leaf tissue) in a drying cabinet (~40°C) and discard the extraction vial cap. Weigh dried leaf tissue after 3 days or after thoroughly driedFor quantification purpose:Place uncapped extraction vials (containing leaf tissue) in a drying cabinet (~40°C) and discard the extraction vial cap. Weigh dried leaf tissue after 3 days or after thoroughly dried
?. [LC-MS metabolite profiling]
To analyze extraction on LC-MS, we use the following LC-MS system and methods:Waters Acquity UPLCWaters Xevo G2-XS QToF mass spectrometerColumn: Ascentis Express C18 HPLC column (10 cm x 2.1 mm, 2.7 µm)Oven temp: 40 °CFlow rate: 0.3 mL/minSolvent A: 10 mM ammonium formate, pH 2.8Solvent B: 100% acetonitrile Table 1. 7-min LC method Table 2. 21-min LC methodColumn: Ascentis Express C18 HPLC column (10 cm x 2.1 mm, 2.7 µm)Oven temp: 40 °CFlow rate: 0.3 mL/minSolvent A: 10 mM ammonium formate, pH 2.8Solvent B: 100% acetonitrile Table 1. 7-min LC method Table 2. 21-min LC methodSolvent A: 10 mM ammonium formate, pH 2.8Solvent B: 100% acetonitrile Table 1. 7-min LC method Table 2. 21-min LC methodTable 1. 7-min LC method Table 2. 21-min LC method  ABC1Time (min)Solvent A %Solvent B %20.0095.05.031.0040.060.045.000.0100.056.000.0100.066.0195.05.077.0095.05.0  ABC1Time (min)Solvent A %Solvent B %20.0095.05.033.0040.060.0415.000.0100.0518.000.0100.0618.0195.05.0721.0095.05.0
To prepare   of 10 mM Ammonium Formate, pH 2.8:
ABC1Time (min)Solvent A %Solvent B %20.0095.05.031.0040.060.045.000.0100.056.000.0100.066.0195.05.077.0095.05.0
ABC1Time (min)Solvent A %Solvent B %20.0095.05.031.0040.060.045.000.0100.056.000.0100.066.0195.05.077.0095.05.0  ABC1Time (min)Solvent A %Solvent B %20.0095.05.033.0040.060.0415.000.0100.0518.000.0100.0618.0195.05.0721.0095.05.0
?. [LC-MS metabolite profiling]
To analyze extraction on LC-MS, we use the following MS settings:For negative ion-mode electrospray ionization:Capillary voltage: 2.00 kVSource temperature: 100°CDesolvation temperature: 350°CDesolvation nitrogen gas flow rate: 600 liters/hCone voltage: 35VMass range: m/z 50 to 1000 with spectra accumulated at 0.1 seconds/functionCollision energies:Function 1: 0VFunction 2: 15VFunction 3: 35VFor positive ion-mode electrospray ionization:Capillary voltage: 3.00 kVSource temperature: 100°CDesolvation temperature: 350°CDesolvation nitrogen gas flow rate: 600 liters/hCone voltage: 35VMass range: m/z 50 to 1000 with spectra accumulated at 0.1 seconds/functionCollision energies:Function 1: 0VFunction 2: 15VFunction 3: 45VFore more detail, please refer to the experiment files:
?. [LC-MS metabolite profiling]
For a list of manuscripts with useful information regarding acylsugar identification (masses, fragmentation patterns and quanitifcation methods), please refer to the description page of this protocol.