Proteomic analysis of the capsaicin response in Arabidopsis roots

To understand the molecular mechanisms involved in response to Capsaicin, we identified the differentially accumulated proteins in response to low capsaicin (0.44 μM) on Arabidopsis seedlings after 48 hours of application using a 2DE proteomic analysis Isoelectric focusing was performed on an Ettan IPGphor 3 electrophoresis unit (Amersham Biosciences) to reach 42000 VH. Following IEF, the strips were equilibrated twice for 20 min in an equilibration solution. For the second dimension, the proteins were separated on 12.5 % SDS polyacrylamide gels, and standard protein markers (Amersham BioScience) were included in the gel. Proteins were stained with Coomassie PhastGel Blue R-350 (GE Healthcare Bio-Sciences AB). Spots with p-value ≤ 0.05 on both PCA and HCA were selected and identified through mass spectrometry. protein spots were analyzed on a nanoflow LC system NanoAcquity (Waters) coupled to a linear ion trap LTQ Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a nanoelectrospray ion source. For the NanoAcquity I system, solvent A consisted of 100 % H2O and 0.1 % formic acid and solvent B consisted of 100 % ACN and 0.1 % formic acid. Peptide samples were prepared as described previously by Shevchenko [24] with some minor modifications. Each reduced and alkylated protein spot was digested with sequencing-grade trypsin (Promega, article number V511A). For the LC-MS/MS experiments, 3-10 μl of trypsin-digested proteins were trapped using pre-column (Symmetry® C18, 5 μm, 180 μm×20 mm, Waters), which was then switched in-line onto a 10-cm capillary UPLC column (100um ID BEH-C18 1.7 um particle size). The column was operated at 35°C. After loading time, the peptides were separated with a 60 min gradient at a 400 nL/min flow rate. The gradient was as follows: 3–50% Solvent B (30 min), 50–85% B (1 min), 85% B (7 min) and 3% B (22 min). Eluted peptides were directly electro-sprayed into the mass spectrometer through a standard coated silica tip (NewObjective, Woburn, MA). The LTQ was operated in data-dependent acquisition mode to automatically alternate between a full scan (m/z 400–2000) and subsequent CID and PQD MS/MS scans in the linear ion trap. CID was performed with helium as collision gas at 40% normalized collision energy and 10 ms of activation time. Data acquisition was controlled by Xcalibur 2.0.7 software (Thermo Fisher Scientific). Tandem mass spectra, obtained from the LTQ-VELOS, were extracted by Proteome Discoverer version 1.2 and searched by use of Sequest against an NCBI RefSeq database with Arabidopsis thaliana (373,345 sequences, February 2024) as a taxonomical option. Precursor mass tolerance was set to ± 2 Da and fragment mass tolerance to 1Da, two incomplete cleavages were allowed. Alkylation of Cys by Carbamidomethylation and oxidation of Met were set as possible modifications. Peptide identification was validated by searching against a decoy database. Theoretical Mm and pI of the identified proteins were predicted using http://web.expasy.org/compute_pi [25]. Identified proteins were used to search homologous by the BLAST protein algorithm against the GenBank non-redundant protein database (Viridiplantae) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (NCBI Resource Coordinators, 2017). Identified proteins were classified into functional categories according to the TAIR Arabidopsis database, UniProt, and KEEG (Kyoto Encyclopedia of Genes and Genomes database (Release 52.0, http://www.genome.jp/kegg). The cellular location of identified proteins was predicted using the public WolfPsort (https://www.genscript.com/wolf-psort.html) [26]. Theoretical Mr and pI of the identified proteins were retrieved from the NCBI RefSeq database.