Proteome discoverer ver 1

Proteins were separated by SDS-PAGE. After aligning of anti-SEPT2 western blot, gel bands corresponding to full length SEPT2 and C-terminal SEPT2 fragment were excised and digested using trypsin then propionic anhydride labeling followed by digestion by either trypsin or Asp-N (Promega). Extracted peptides were analyzed by LC-MS/MS (EasyLC 1200 coupled to Fusion Lumos, Thermo Scientific), separated by reversed phase with a gradient increasing from 1% B to 40% B in 40 min. MS data were queried against Uniprots Human database (March 2016) using no enzymatic (Asp-N experiment) or semi-tryptic constraints using Proteome Discoverer ver1.4 (Thermo Fisher)/Mascot 2.4 (Matrix Science). Oxidation of methionine was allowed. For samples treated with propionic anhydride, propionylation of N-termini and lysine were allowed as variable modifications. Matched peptides were filtered using < 5% FDR (Percolator 2) and mass accuracy < 5 ppm in addition to manual validation of tandem spectra. Extracted areas of Septin-2 peptides obtained by the three different digestion strategies for intact SEPT2 and C-terminal SEPT2 were summed per residue and plotted as a function of SEPT2 using PepEx 3, after normalization to the signal of the most C-terminal residues.