iTRAQ Proteomic Analysis of Wheat (Triticum aestivum L.) Genotypes Differing in Waterlogging Tolerance
Strong cation exchange (SCX) fractionation and LC-MS/MS analysis The combined labeled samples were bound to a SCX fractionation column connected with a high performance liquid chromatography (HPLC) system. The peptide mixture was re-dissolved in the buffer A (20 mM ammonium formate in water, pH10.0), and then fractionated by high pH separation using Ultimate 3000 system (Thermo Fisher scientific, MA, USA) connected to a reverse phase column (Gemini-NX 3u C18 110A column, 2.0 mm x 150 mm, 3 μm, (Waters Corporation, MA, USA). High pH separation was performed using a linear gradient starting from 5% to 45% buffer B (20 mM ammonium formate in 80% ACN, pH 10.0) in 40 min. The column flow rate was maintained at 0.2 mL/min and column temperature was maintained at 30℃. A total of 12 fractions were collected, and each fraction was dried in a vacuum concentrator for the next step. Peptide fractions were resuspended with 30 μL solvent C ( water with 0.1% formic acid), respectively, and separated by nanoLC and analyzed by electrospray tandem mass spectrometry. The experiments were performed on an Easy-nLC 1000 system (Thermo Fisher Scientific, MA, USA). A total of 10 μL peptide sample was loaded onto the trap column (Thermo Scientific Acclaim PepMap C18, 100 μm × 2 cm), with a flow of 10 μL/min for 3 min and subsequently separated on the analytical column (Acclaim PepMap C18, 75 μm × 15 cm) with a linear gradient, from 3% to 32% solvent D (ACN with 0.1% formic acid) in 120 min. The column flow rate was maintained at 300 nL/min. The fusion mass spectrometer was run in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra (m/z 350-1550) were acquired with a mass resolution of 120 K, followed by sequential high energy collisional dissociation MS/MS scans with a resolution of 30 K. The isolation window was set as 1.6 Da. MS/MS fixed first mass was set at 110. In all cases, one microscan was recorded using dynamic exclusion of 45 seconds. Database search and Quantification The mass spectrometry data were transformed into MGF (Mascot generic format) files with Proteome Discovery 1.2 (Thermo, PA, USA) and analyzed using Mascot software version 2.3.2 (Matrix Science, London, UK). Mascot database was set up for protein identification using Triticum aestivum L database in NCBI nr (release 2017_03); SwissProt/UniprotKB (release 2018_06) and International Protein Index (IPI; version 3.16). Trypsin/P was chosen as the enzyme with two missed cleavages allowed; Peptide tolerance was set at 10 ppm, and Mascot was searched with a fragment ion mass tolerance of 0.050 Da; a parent ion tolerance of 10.0 PPM. Significance threshold p < 0.05 (with 95% confidence). The average values of the biological replicates were used to indicate the final protein abundances for each sample. Proteins with a 1.2-fold change between samples and a p value less than 0.05 were determined as differentially expressed proteins (DEPs).