2.5  Protein Extraction, Digestion, and iTRAQ Labeling
At 24 h postinfection (hpi), both PDCoV- and mock-infected IPEC-J2 cells grown in T25 flasks (∼5 × 106 cells/flask) were rinsed twice with prechilled PBS, and then harvested with disposable cell scrapers. Three flasks of each group (PDCoV or mock) were harvested and used as three independent biological replicates. After centrifugation at 300g for 10 min, the cell pellets from each flask were lysed with 800 μL of RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% SDS, 8 M urea and protease inhibitors (Beyotime). The cell lysates were further sonicated on ice for 5 min with 10 s bursts and 10 s pauses between cycles. After centrifugation for 20 min at 12 000g and 4 °C, the supernatant was collected and used as the total cellular proteins. The concentration of protein samples was determined using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA).
The total cellular proteins from each biological replicate were equally divided into three aliquots, which were used as three independent technical replicates for the LC-MS/MS runs. For tryptic digestion and iTRAQ labeling, an aliquot of each protein sample containing ∼100 μg of total cellular proteins was adjusted to a 100-μL final volume using the RIPA lysis buffer. A final concentration of 10 mM of tris (2-carboxyethyl) phosphine (TCEP) was added to each protein sample, which was then incubated at 37 °C for 1 h. Afterward, iodoacetamide was added at a final concentration of 40 mM, and the protein solution was incubated for 40 min at room temperature shielded from light. Subsequently, prechilled acetone was added to the protein solution in a ratio of 6:1 and precipitated at −20 °C for 4 h. After centrifugation (10 000g, 4 °C) for 20 min, the precipitate was dissolved with 100 μL of 100 mM triethylammonium bicarbonate (TEAB). The processed protein samples were digested with 2 μg/μL of trypsin overnight at 37 °C. Following tryptic digestion, the generated peptides were dried by vacuum centrifugation, and redissolved with 20 μL of 0.5 M TEAB. The prepared peptides were then labeled with an iTRAQ reagents-8 plex kit (AB Sciex, Foster City, CA, USA) as per the manufacturer’s protocol. Briefly, the three independent biological replicates of mock-infected cellular samples were each labeled with iTRAQ-113, iTRAQ-114, and iTRAQ-115; the three independent biological replicates of PDCoV-infected samples were each labeled with iTRAQ-116, iTRAQ-119, and iTRAQ-121. All the labeled samples of each group were mixed with an equal amount, and then fractionated using an ACQUITY ultra performance liquid chromatography (UPLC) system (Waters Corp., Milford, MA, USA) combined with an ACQUITY UPLC BEH C18 column (300 Å, 1.7 μm, 2.1 mm × 150 mm). Finally, a total of 10 fractions of each group were collected. After merging two fractions of each group into one, the pooled 10 fractions were dried by using a rotary vacuum concentrator.