Ltq lc ms ms

Co-IP samples of sGFP and CjWRKY1-sGFP proteins were obtained as described above. Co-immunoprecipitated samples were separated by SDS-PAGE and silver stained (Supplementary Fig. S2). Protein bands were extracted, and proteins were digested in-gel with MS grade modified trypsin (Promega). The extracted peptides were subjected to LTQ LC/MS/MS (Thermo Fisher Scientific). The collected ion spectrum data were analysed with the help of Mr. Y. Watanabe (Proteomic Facility Lab., Grad. Sch. Biostudies, Kyoto Univ.) using MassMatrix version 2.4.2 (www.massmatrix.net/)38 (link).

Extracted peptides were subjected to nanoLC-ESI-MS/MS mass spectrometry (LTQ-LC-MS/MS, Thermo Fisher Scientific) using an EasyNano LC system equipped with a 15-cm ODS column (NTCC-360/75-3-125 C18, particle diameter 3 μm, 0.075 mm × 125 mm; Nikkyo Technos, Tokyo, Japan). The flow rate was adjusted to 300 nL/min for all analyses. The raw data files were processed using software MaxQuant combined with Andromeda (Max Planck Institute of Biochemistry, Bavaria, Germany) for peptide searching, and label-free quantification was carried out using Perseus software (version 1.6.0.7)^{44 (link),45 (link)}. Peptide precursor mass tolerance was set at 10 ppm, and MS/MS tolerance was set at 0.5 Da. Search criteria included carbamidomethylation of cysteine as a fixed modification and oxidation of methionine (+15.9949) as a variable modification. Searches were performed with full tryptic digestion and a maximum of 2 missed cleavages was allowed. The reverse database search option was enabled, and all peptide data was filtered to satisfy a false discovery rate (FDR) of 1%.