Trypsin

iTRAQ analysis was implemented at BGI (Shenzhen, China). Total protein was extracted from seedling roots under the treatment of CK and T according to the company’s method [93 (link),94 (link)]. The protein concentration was determined using the Bradford dye-binding assay [95 (link)]. For each sample, the solution containing 100 µg of protein was transferred to a new tube. trypsin Gold (Promega, Madison, WI, USA) with the ratio of protein:trypsin = 40:1 was added and hydrolyzed at 37 °C for 4 h. Then, trypsin Gold was added once again with a ratio of protein:trypsin = 40:1, and digested for 8 h at 37 °C. After trypsin digestion, desalination was carried out with the StrataXC18 column (Phenomenex, Torrance, CA, USA), and vacuum drying was carried out according to the manufacturer’s protocol. These peptides are dissolved in 0.5M TEAB by vortex. When the iTRAQ labeling reagent reaches room temperature, it is transferred and combined with the appropriate sample. The peptides from 8723 and P138 were labeled according to the manufacturer’s protocol for 8-plex iTRAQ reagent (Applied Biosystems, Foster city, CA, USA). The iTRAQ tag is shown in Table S8.

The proteins were digested with trypsin as previously described [37 (link)]. In detail, the protein sample was diluted after adding 100 mM NH₄CO₃ to a urea concentration of less than 2 M. Then, trypsin (Promega, Madison, CT, USA) was added at 1:50 trypsin-to-protein mass ratio for the first digestion at 37 °C overnight and a 1:100 trypsin-to-protein mass ratio for a second four-hour digestion. After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex, Torrance, CA, USA) and vacuum-dried. Peptide was reconstituted in 0.1 M NaAc (pH = 5.99). The samples are differentially isotope labeled in parallel in different tubes by adding four µL CH₂O or CD₂O to the samples to be labeled with light (control Cell) and heavy (viral infected cell) dimethyl, respectively. The reactions were mixed briefly and added four µL of 0.6 M NaBH₃CN, incubated in a fume hood for one hour at room temperature. Finally, the labeling reactions were quenched by adding 16 µL of 1% ammonia solution, then adding formic acid for desalted and dried by vacuum centrifugation. Then the tryptic peptides were subjected to lysine acetylated peptide enrichment using a previously described method [66 (link)]. The resulting peptides were cleaned with C18 ZipTips (Merck Millipore) according to the manufacturer’s instructions, followed by LC–MS/MS analysis.

The liver samples were ground and then sonicated on ice 3 times by a high-intensity sonicator (Scientz, Ningbo, China) in lysis buffer (3 µM TSA, 50 mM NAM, 0.1% protease inhibitor cocktail, 10 mM DTT, 8 M urea). Centrifugation was performed for 10 min at 20,000× g and 4 °C to remove residual debris. Subsequently, the protein was deposited and the supernatant was discarded by centrifugation. The precipitate was washed and re-dissolved in buffer (100 mM TEAB, 8 M urea, pH 8.0). The concentration of protein was determined by the 2-D Quant kit. The protein solution was reduced by 10 mM DTT and alkylated by 20 mM IAA (Sigma, St. Louis, MO, USA) for digestion. An amount of 100 mM TEAB was added to less than 2 M in urea concentration to dilute the protein samples. Trypsin (Promega) was added for the first digestion with a 1:50 mass ratio of Trypsin-to-protein and with a 1:100 mass ratio for the second 4-hour-digestion. The iTRAQ labeling and analysis were implemented by Novogene Bioinformatics Technology Co. Ltd. (Beijing, China). Peptides were desalted by Strata × C18 SPE column (Phenomenex, Torrance, CA, USA) and vacuum-dried after Trypsin digestion. Peptides were reconstituted in 0.5 M TEAB and processed by a 6-plex TMT kit (Thermo, Waltham, MA, USA). The mixtures were subsequently incubated at room temperature for 2 h and combined, desalted, and lyophilized.

Dithiothreitol (5 mM, catalog no. D9163, Sigma-Aldrich, USA) was added to the protein solution, and reduction was allowed to proceed at 56°C for 30 min. The protein solution was then incubated with 11 mM iodoacetamide (catalog no. V900335, Sigma-Aldrich, USA) at room temperature in darkness for 15 min. The urea concentration of the solution was then diluted to <2 M using 100 mM tetraethylammonium bromide (catalog no. 17902, Sigma-Aldrich, USA). Finally, trypsin (catalog no. V5111, Promega, USA) was added at a trypsin-to-protein mass ratio of 1:50 for the first round of digestion for overnight and 1:100 for the second round of digestion for 4 h. Peptides were desalted on a Strata X C18 SPE column (Phenomenex, USA) and vacuum dried after trypsin digestion. They were then labeled using a TMT 10-plex isobaric label kit (catalog no. 90111, Thermo, USA), according to manufacturer instructions.

Proteins (100 µg) were mixed with 10 mM DTT and incubated at 37 °C for one hour, and then 55 mM iodoacetamide was added to alkylate at room temperature for one hour in the dark. After reduction and alkylation, 2 μg of trypsin (Promega, USA) was added at a protein/trypsin ratio of 50:1, and then digested at 37 °C for 12 h, followed by centrifugation at 12,000× rpm for 15 min. After trypsin digestion, acidulation was conducted by adding an equal volume of 0.1% formic acid, then purified by a Strata-X C18 column (Phenomenex, 8B-S100-UBJ) for three times, washed twice with 0.1% formic acid + 5% acetonitrile, and eluted with 0.1% formic acid + 80% acetonitrile. Peptides were evaporated to dryness by vacuum centrifugation, and then the dried peptides were reconstituted in 0.5 M TEAB solution (pH 8.5).
According to the manufacturer’s protocol, proteins were labeled with an 8-plex iTRAQ Reagent Multiplex Kit (AB Sciex, USA). For labeling, one unit of iTRAQ reagent was thawed and reconstituted in 24 μL of isopropanol, and proteins were labeled with isobaric tags as follows: 113- and 114-tag for C1, 115- and 116-tag for C2, 117- and 118-tag for M200, and 119- and 121-tag for M1000. The labeled samples were incubated at 25 °C for 1 h, and the reaction was stopped with 100 μL of ddH₂O. Then, the differentially labeled peptide mixtures were pooled and dried by vacuum centrifugation.

Proteins were reduced in 5 mM dithiothreitol at 55 °C for 30 min, alkylated in 10 mM iodoacetamide for 15 min at room temperature in the dark, precipitated in pre-cold acetone at −20 °C overnight, and diluted by adding 200 mM TEAB. Next, a first trypsin (MS grade; Corning, Somerville, MA, USA) digestion step was performed overnight at a 1:50 trypsin-to-protein mass ratio, and a second digestion was then conducted at a 1:100 ratio for 4 h. After the digestion steps, peptides were desalted on a Strata X C18 SPE column (Phenomenex, Torrance, CA, USA) and dried under vacuum. A peptide mixture was reconstituted in 100 mM TEAB from the TMT kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Samples were then incubated for 1 h at room temperature and dried under vacuum centrifugation, the reactions were terminated with 5% hydroxylamine addition, and the samples were lyophilized and then finally stored at −80 °C [23 (link)].