Q exactive hf mass spectrometry system

About 10-day-old OE-ZmBSK1 transgenic lines were treated with 200 mM NaCl for 0 or 10 h, and then total proteins from the leaves were extracted using lysis buffer [10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5 mM DTT, and protease inhibitor cocktail]. Protein extracts were immunoprecipitated with anti-Flag antibody (1:300, Abmart) bound to protein A/G agarose beads in PBS buffer containing protease inhibitor cocktail for 8 h at 4°C. The beads were washed for three times with PBS buffer and were boiled in SDS loading buffer (Beyotime, China). After centrifugation, the supernatants were processed according to a previous publication (Wang et al., 2015 (link)), and were analyzed using EASY-nLC 1200 ultra-high performance liquid system (Thermo Scientific, United States) and Q Exactive HF mass spectrometry system (Thermo Scientific, United States). For protein identification, the MS data were searched against maize database in UniProtKB using the Paragon algorithm in ProteinPilot ™ Software (v5.0.2, SCIEX). For the identified proteins, selected certain filtering criteria and peptides with an unused score > 1.3 were considered as credible peptides, and proteins containing at least one unique peptide were retained.