DOI: 10.25345/c5ws8hx96
title: MassIVE MSV000095072 - Single-cell proteomics of Arabidopsis leaf mesophyll protoplasts in the context of drought stress
title: We followed the Arabidopsis tape-sandwich protocol to isolate the leaf protoplast enriched with palisade mesophyll cells. Briefly, control and WD-stressed Arabidopsis rosette leaves were placed on 3M tape, and abaxial cell layers were peeled off by gently removing the tape, thereby exposing the adaxial side with enriched palisade mesophyll cells. The exposed leaf (n=6) from control and WD stress conditions was placed in a petri plate, and 20 mL of protoplasting enzyme solution was added. The enzyme solution contained 1% cellulase (CELLULYSIN) and 0.25% macerozyme R10 dissolved in a buffer made of 0.4M mannitol, 20 mM potassium chloride, 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer and adjusted to pH 5.7. The enzyme solution was heated at 55 deg C for 10 mins to dissolve the enzymes into the buffer followed by cooling the solution on ice for 5 minutes. Following that, 10 mM calcium chloride was added to the enzyme solution, and it was used for protoplast isolation. The leaf tissue was incubated with the enzyme solution for 45 minutes and kept on a rotatory shaker maintained at 150 rpm at 25 deg C in the dark to allow the enzymes to digest the cell walls to liberate the protoplasts. Three batches of protoplast isolation were performed from control and WD-stressed leaf tissue to yield sufficient protoplasts for single-cell proteomics workflow. After enzymatic digestion, the protoplast solution was carefully transferred to 50 mL falcon tubes and centrifuged at 13 G at room temperature using an IEC Centra MP4R refrigerated centrifuge. The protoplast pellet was gently washed two times using the same buffer used to dissolve enzymes (without mannitol) and filtered using a 0.45 um filter to remove the cell debris. The filtered leaf protoplasts were counted using a hemocytometer then immediately sorted using the cellenONE. We employed an in-house assembled nanoPOTS autosampler for LC-MS/MS analysis. The autosampler contains a custom packed SPE column (100 um i.d., 4 cm, 5 um particle size, 300 A pore size C18 material, Phenomenex) and analytical LC column (50 um i.d., 25 cm long, 1.7 um particle size, 190 A pore size C18 material, Waters) with a self-pack picofrit (cat. no. PF360-50-10-N-5, New Objective, Littleton, MA). The analytical column was heated to 50 deg C using an AgileSleeve column heater (Analytical Sales and services, Inc., Flanders, NJ). Briefly, samples were dissolved with Buffer A (0.1% formic acid in water) on the chip, then trapped on the SPE column for 5 min. After washing the peptides, samples were eluted at 100 nL/min and separated using a 30 min gradient from 8% to 35% Buffer B (0.1% formic acid in acetonitrile). An Orbitrap Lumos Tribrid MS (ThermoFisher Scientific) with FAIMS, operated in data-dependent acquisition mode, was used for all analyses. Source settings included a spray voltage of 2,400 V, ion transfer tube temperature of 200 deg C, and carrier gas flow of 4.6 L/min. For TIFF method16 samples, ionized peptides were fractionated by the FAIMS interface using internal compensation voltage stepping (-45, -60, and -75 V) with a total cycle time of 0.8 s per compensation voltage. Fractionated ions within a mass range 350-1600 m/z were acquired at 120,000 resolution with a max injection time of 500 ms, AGC target of 1E6, RF lens of 30%. Tandem mass spectra were collected in the ion trap with an AGC target of 20,000, a "rapid" ion trap scan rate, an isolation window of 1.4 m/z, a maximum injection time of 120 ms, and a HCD collision energy of 30%. For the TIFF library samples, a single compensation voltage was used for each LC-MS run with slight modifications to the above method where cycle time was increased to 2 s and maximum injection time was set to 118 ms. Precursor ions with a minimum intensity of 1E4 were selected for fragmentation by 30% HCD and scanned in an ion trap with an AGC of 2E4 and an IT of 150 ms. Precursor ions with intensities &gt; 1E4 were fragmented by 30% HCD and scanned with an AGC of 2E4 and an IT of 254 ms. Downstream analysis was performed in FragPipe.