Acetonitrile

Protein spots to be identified were manually excised from Coomassie stained SDS-PAGE. Excised gel fractions were washed in 100 mM ammonium bicarbonate (Sigma-Aldrich)/acetonitrile (Merck) (1:1, v/v) until completely destained. After drying with acetonitrile, gel fragments were rehydrated in 20 μl of trypsin solution (Trypsin Gold, Mass Spectrometry grade, Promega) at 13 ng/μl in 10 mM ammonium bicarbonate solution containing 10% acetonitrile (v/v). Then, digestion was performed at 37°C for 16 h. Peptide extraction was carried out twice for 1 h with 20 μl 50% acetonitrile/5% formic acid (Sigma-Aldrich). In-gel trypsin digestion products were analyzed by a Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometer (MALDI-TOF/TOF LIFT, AutoFlex III, Bruker Daltonics) in positive/reflector mode controlled by FlexControl software Version 3.3, as previously described (Barbey et al., 2012 (link)).

The proteins that were purified by GFP-Trap were separated by 12% SDS-PAGE and stained with Oriole Fluorescent Gel Stain (Bio-Rad, Hercules, CA, USA) for 90 min. The stained protein bands were excised from the gels, washed twice with 100 mM of NH₄HCO₃ containing 30% acetonitrile (Wako, Osaka, Japan), washed with 100% acetonitrile, and then dried in a vacuum concentrator. The gels were reductively alkylated with 10 mM of dithiothreitol (DTT) in 100 mM of NH₄HCO₃ for 60 min at 56 °C and with iodoacetamide in 100 mM of NH₄HCO₃ for 30 min at 25 °C. The gels were washed with 100 μL of 100-mM NH₄HCO₃ for 10 min and dehydrated through the addition of acetonitrile. The dried gels were treated with 2 μL of 0.5 μg/μL of trypsin (Promega, Madison, WI, USA) in 50 mM of NH₄HCO₃ and incubated at 37 °C for 12 h. Peptides were extracted with 50 mM of NH₄HCO₃ in 1% trifluoroacetic acid and 50% acetonitrile. Sample preparation for MALDI analysis was according to the method of Shevchenko et al. [64 (link)]. Peptides of proteins that associate with OsVQ13 were identified through MALDI-TOF MS analysis with a Voyager-DE STR (Thermo Fisher Scientific, Waltham, MA, USA). Experiments using OsVQ13GFP-overexpressing rice plants were repeated five times, and four out of the five stained gels are presented in the Supplementary Figure S1.

TMT10plex Isobaric Label Reagent Set, Pierce Quantitative Colorimetric Peptide Assay (Thermo Fisher Science, Waltham, MA, USA). Triethylammonium bicarbonate buffer (1.0 M, pH 8.5 ± 0.1), Tris (2-carboxyethyl) phosphine hydrochloride solution (0.5 M, pH 7.0), iodoacetamide (IAA), formic acid (FA), acetonitrile (MeCN), methanol (MeOH), and trypsin from bovine pancreas (Promega, Madison, WI, USA) were used. Ultrapure water was obtained from a Millipore purification system.