DOI: 10.25345/c5pv6bm6q
title: MassIVE MSV000100432 - Spatially resolved proteome profiling of switchgrass root-tips under well-watered and drought-stressed field conditions using LCM coupled with nanoPOTS proteomics
title: Mass spectrometry-based proteomic datasets were generated from Switchgrass 'Alamo' plants 2015 at the Agricultural Research and Education Experimental Station, Tennessee State University, Nashville, Tennessee under severe drought-stressed conditions in Summer 2022. Roots were excavated to a depth of 40 cm, and the soil on the root surface was analyzed for water content. Three biological replicates, each comprising 10 plants/plot, were dug for each of the two treatment conditions. Root tips were collected and immediately submerged in prechilled fixative solution containing ethanol and acetic acid (3:1). Tissue fixation and embedding followed the protocol of Balasubramanian et al. (2021) prior to laser-capture microdissection (LCM) of root-tip specific cell types on PEN membrane slides. LCM-isolated material was collected on microPOTS chips containing DMSO droplets for efficient capturing. For spatially-resolved proteomics with microPOTS, the samples were placed uncovered, upside down in a 70 C incubator until all DMSO droplets were evaporated. Next, 1.6 uL of cell lysis buffer (0.2% (w/v) DDM, 1X phosphate-buffered saline, 1 mM DTT, and 50 mM ammonium bicarbonate was aliquoted into each microwell. The chip was covered with a sterile glass slide and secured within a humidity chamber and incubated at 70 C for one hour. Using in-house nanosampling robot, 200nL of 18mM of IAA in 50 mM ammonium bicarbonate was dispensed into each well. The samples were left to incubate within a humidity chamber, in the dark, at room temperature for 30 minutes. Next, the samples were digested by dispensing 200nL of 4 ng of Lys-C (MS grade, Promega) and 200nl of 8 ng of Trypsin (Promega) in 50mM ammonium bicarbonate, placed in humidity chamber, and incubated for 10 hours at 37 C. To quench digestion, 100nL of 5% formic acid (FA) was dispensed into each well and left to incubate at room temperature for approximately 15 min. The samples were then removed from incubation chamber and placed uncovered within a vacuum desiccator until all droplets had evaporated. The prepped samples were stored at -20 C until analysis. For LC-MS/MS, we employed an in-house assembled microPOTS 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 Angstrom pore size C18 material, Phenomenex) and analytical LC column (50 um i.d., 25 cm long, 1.7 um particle size, 190 Angstrom pore size C18 material, Waters) with a self-pack picofrit. The analytical column was heated to 50 C using AgileSleeve column heater. 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 60 min gradient from 8% to 35% Buffer B (0.1% formic acid in acetonitrile). For mass spectrometry analysis, an Orbitrap Lumos Tribrid mass spectrometer with high field asymmetric-waveform ion mobility spectrometry (FAIMS) was operated in data-dependent acquisition mode. Source settings included a spray voltage of 2,400 V, ion transfer tube temperature of 200 C, and carrier gas flow of 3.5 L/min. 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 350-1500 m/z were acquired at 120,000 resolution with a max injection time of 118 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 180 ms, and an HCD collision energy of 30%. For database searching, all proteomic data raw files were processed by FragPipe (version 22.0) and searched against the draft Panicum virgatum protein sequence assembly (version 5.1) acquired from the Joint Genome Institute. This database included target sequences as well as decoy sequences and common protein contaminants (260,120 forward and reversed entries accessed June 2024). MSFragger version 4.1, IonQuant version 1.10.27, and Philosopher version 5.1.1 were applied to the search. Search settings included a precursor mass tolerance of +/-20 ppm, fragment mass tolerance of +/-250 ppm, deisotoping, carbamidomethylation as a fixed modification, and several variable modifications (oxidation of methionine and N-terminal acetylation). Protein and peptide identifications were filtered to a false discovery rate of less than 1% within FragPipe. IonQuant match-between-runs (MBR) was set to "True" and an MBR FDR of 2% at ion level was used to reduce false matching.