RATIONALE: Proteins undergo post-translational modifications and proteolytic processing that can affect their biological function. Processing often involves the loss of single residues. Cleavage of signal peptides from the N-terminus is commonly associated with translocation. Recent reports have suggested that other processing sites also exist. METHODS: The secreted proteins from S. aureus N315 were precipitated with trichloroacetic acid (TCA) and amidinated with S-methyl thioacetimidate (SMTA). Amidinated proteins were digested with trypsin and analyzed with a high-resolution orbitrap mass spectrometer. RESULTS: Sixteen examples of Staphylococcus aureus secretory proteins that lose an N-terminal signal peptide during their export were identified using this amidination approach. The N-termini of proteins with and without methionine were identified. Unanticipated protein cleavages due to sortase and an unknown protease were also uncovered. CONCLUSIONS: A simple N-terminal amidination based mass spectrometry approach is described that facilitates identification of the N-terminus of a mature protein and the discovery of unexpected processing sites.
RATIONALE: Proteins undergo post-translational modifications and proteolytic processing that can affect their biological function. Processing often involves the loss of single residues. Cleavage of signal peptides from the N-terminus is commonly associated with translocation. Recent reports have suggested that other processing sites also exist. METHODS: The secreted proteins from S. aureus N315 were precipitated with trichloroacetic acid (TCA) and amidinated with S-methyl thioacetimidate (SMTA). Amidinated proteins were digested with trypsin and analyzed with a high-resolution orbitrap mass spectrometer. RESULTS: Sixteen examples of Staphylococcus aureus secretory proteins that lose an N-terminal signal peptide during their export were identified using this amidination approach. The N-termini of proteins with and without methionine were identified. Unanticipated protein cleavages due to sortase and an unknown protease were also uncovered. CONCLUSIONS: A simple N-terminal amidination based mass spectrometry approach is described that facilitates identification of the N-terminus of a mature protein and the discovery of unexpected processing sites.
Authors: Harold Tjalsma; Haike Antelmann; Jan D H Jongbloed; Peter G Braun; Elise Darmon; Ronald Dorenbos; Jean-Yves F Dubois; Helga Westers; Geeske Zanen; Wim J Quax; Oscar P Kuipers; Sierd Bron; Michael Hecker; Jan Maarten van Dijl Journal: Microbiol Mol Biol Rev Date: 2004-06 Impact factor: 11.056
Authors: Celia R W Carneiro; Edilberto Postol; Regina Nomizo; Luiz F L Reis; Ricardo R Brentani Journal: Microbes Infect Date: 2004-05 Impact factor: 2.700