Dissolution buffer

Lysis solution [9 M urea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate, 1% dithiothreitol, and 1% immobilized pH gradient buffer (GE Healthcare)] was used to extract protein from each sample. The mixture was centrifuged at 15 000 rpm for 15 min after 1 h of incubation at 30 °C. Dissolution buffer (AB Sciex, Foster City, CA, USA) was used to dissolve the total protein from each sample, and each sample was labeled using an iTRAQ Reagent-8 plex Multiplex Kit (AB Sciex). The labeled sample was separated by liquid chromatography (LC) with an Eksigent nanoLC-Ultra 2D system (AB SCIEX).
LC fractions were analyzed with a Triple TOF 5600 mass spectrometer (AB SCIEX) under the following conditions: ion spray voltage, 2.5 kV; nebulizer gas pressure, 5 PSI; curtain gas pressure, 30 PSI; and interface heater temperature, 150 °C. The information-dependent acquisition mode was applied for 35 product ion scans (2⁺ to 5⁺) above a threshold ion count of 150 in the mass spectrometry survey scan. The dynamic exclusion duration was 18 s. The data for iTRAQ protein were analyzed using Protein Pilot Software Version 4.0 against the database Uniprot_grape.

Protein solution (30 μl) was taken and DTT was added to a 10 mM final concentration, and boiled for 5 min, and then cooled to room temperature. UA buffer (200 μl; 8 M urea, 150 mM Tris-HCl, pH 8.0) was added and centrifuged at 14 000 g for 15 min for two times. UA buffer (100 μl; 100 mM iodoacetamide in UA) was added by vortex at 600 r.p.m. for 1 min. The samples were incubated for 30 min in darkness, and centrifuged at 14 000 g for 15 min. Dissolution buffer (100 μl; AB SCIEX, Foster City, CA, USA; DS buffer) was added and centrifuged at 14 000 g for 15 min for two times. Proteins for each sample were incorporated into 30 μl SDT buffer. Then, 100 μl iodoacetamide (100 mM indole-3-acetic acid in UA buffer) was added to block reduced cysteine residues. Finally, the protein suspensions were digested with 4 μg Trypsin (Promega, Madison, WI, USA) in 40 μl DS buffer overnight at 37 °C, and the resulting peptides were collected as a filtrate.^{23 (link)} The filtrated peptides of each sample were desalted on C18 Cartridges (Empore SPE Cartridges C18, bed I.D. 7 mm, volume 3 mL, Sigma, St Louis, MO, USA), concentrated by vacuum centrifugation and reconstituted in 40 μL of 0.1% (v/v) formic acid. The peptide content was estimated by ultraviolet light spectral density at 280 nm using an extinction coefficient of 1.1 of 0.1% (g L⁻¹) solution.

Equal amount of the sample protein extracts were added to 100 mM DTT (Bio-Rad) and subjected to water bath at 90 °C for 5 min, and then cool to room temperature. Followed by the addition of 200 μL UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0) to the sample and mix. The sample was transferred into a 30kd ultrafiltration centrifuge tube and centrifuged at 14000 × g for 15 min. Then added 200 μL UA buffer to the sample and centrifuged at 14000 × g for 15 min. The supernatant obtained was mixed with 100 μL of iodoactamide (50 mM iodoactamide in UA buffer) and shaken (600 rpm) for 1 min. Then samples were incubated at room temperature for 30 min in dark. Next, 100 μL of UA buffer was added to the sample and centrifuged at 14000 × g for 10 min and repeated this process twice. Subsequently, 100 μL of dissolution buffer (AB SCIEX) was added to the sample and centrifuged at 14000 × g for 10 min, and repeated this process twice. The supernatant was discarded and precipitate was re-dissolved in 40 μL of trypsin buffer (3 μg Trypsin in 40 μL dissolution buffer), shaking at 600 rpm for 1 min followed by enzymatic hydrolysis at 37 °C for 16–18 h. The enzymatic hydrolysis peptides were collected by centrifuged at 14000 × g for 10 min. Finally, Optimal density of the enzymatic hydrolysis peptides were determined at a wavelength of 280 nm using a micro-plate reader (Biotek, Winooski, VT, USA).