Proteomic analysis of S. elongatus

Total proteins from S. elongatus were extracted, as described [22 (link)]. Total protein extracts (4 mg/IP) were immunopurified using anti-GFP antibody-coupled magnetic beads of 50 nm (MACS^® Technology, Miltenyi, Bergisch Gladbach, Germany), digested in column with trypsin and analyzed in a single run on the mass spectrometer [23 (link)]. The resulting tryptic peptide mixture was desalted prior to LC-MS/MS analysis on a C18 ZipTip (Omix C18 100 μL tips, Varian, Santa Clara, CA, USA), and the purified peptide mixture was analyzed by LC-MS/MS using a nanoflow RP-HPLC (LC program: linear gradient of 3–40% B in 100 min, solvent A: 0.1% formic acid in water, solvent B: 0.1% formic acid in acetonitrile), which was on-line coupled to a linear ion trap Orbitrap (Orbitrap-Fusion Lumos, Thermo Fisher Scientific, Waltham, MA, USA) mass spectrometer operating in positive ion mode. Data acquisition was carried out in a data-dependent fashion, and the 20 most abundant, multiply charged ions were selected from each MS survey for MS/MS analysis (MS spectra were acquired in the Orbitrap, and CID spectra were acquired in the linear ion trap).