Arabidopsis thaliana N-terminal proteome,LC-MS

An extended ChaFRADIC workflow was applied to analyze the N-terminal proteome of Arabidopsis thaliana seedlings. Using iTRAQ protein labeling, a multi-enzyme digestion approach including trypsin, GluC, and subtilisin, a total of 200 μg per enzyme were used, and only 1/3 of each ChaFRADIC-enriched fraction were analyzed by LC-MS. Furthermore, our goal was to gain insights of the Met-excision dogma where initiator Met residues are cleaved posstranslationally if the second residue is small, as well as the N-end rule degradation pathway (NERD) discriminating between stabilizing/destabilizing functions of N-terminal amino acid residues in Arabidopsis. We found bona fide NERD destabilizing residues underrepresented. The list of neo N-termini from wild type samples represents an extremely helpful resource for excluding pseudo-candidates of NERD.