Pierce c 18 spin columns

The sample collected at the end of ultrafiltration and before cryoprotectant addition (1 mL) were concentrated to 50 µL using a SpeedVac system (Savant Instruments, Farmingdale, NY, USA) and treated with RapiGest SF (Waters Corporation, Milford, MA, USA) at the final concentration of 0.25% (w/v). After incubation at 100 °C for 20 min, the samples were cooled at room temperature and centrifuged at 2200× g for 10 min. Subsequently, protein concentration was assayed using the SPNTM Protein Assay kit (G-Biosciences, St. Louis, MO, USA) and 50 ± 0.5 μg of protein from each sample was digested with Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA) using a 1:50 (w/w) enzyme/substrate ratio at 37 °C overnight. The next morning, an additional aliquot of enzyme was added (enzyme/substrate ratio of 1:100 (w/w)). The enzymatic reaction was chemically stopped after 4 h by acidification with TFA 0.5% (Sigma-Aldrich Inc., St. Louis, MO, USA), incubation at 37 °C for 45 min and centrifugation at 13,000× g for 10 min in order to remove hydrolytic RapiGest SF by-products. Finally, the samples were desalted by PierceTM C-18 spin columns (Thermo Fisher Scientific, Waltham, MA, USA), concentrated in a SpeedVac (Savant Instruments, Farmingdale, NY, USA) at 60 °C and resuspended in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) at a concentration of 0.1 µg/µL.

Liquid-nitrogen-frozen leaf and root tissues (about 100 mg) were ground to a fine powder with a pestle and mortar, and membrane proteins were then extracted using a membrane extraction kit according to the manufacturer’s protocol using plasma membrane extraction protocols (Cat#SM-005-P; Invent Biotechnologies, Plymouth, MN, USA). The extracted proteins were dissolved in 8 M urea buffer for further digestion and sample preparation.
Digestion of proteins was performed using SMART digest^TM trypsin kit (60109-101; Thermo Scientific, Waltham, MA, USA) in solution. Protein reduction and alkylation were achieved with 10 mM dithiothreitol at 56 °C for 30 min followed by 25 mM iodoacetamide at room temperature for 25 min. The digested peptides were purified using Pierce^TM C-18 spin columns (Thermo Scientific, Waltham, MA, USA) and finally dissolved in 0.1% formic acid (FA).