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Identification of N‐terminal protein processing sites by chemical labeling mass spectrometry
Author(s) -
Misal Santosh A.,
Li Sujun,
Tang Haixu,
Radivojac Predrag,
Reilly James P.
Publication year - 2019
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.8435
Subject(s) - chemistry , orbitrap , mass spectrometry , bottom up proteomics , biochemistry , proteolysis , protease , tandem mass spectrometry , signal peptide , cleavage (geology) , trypsin , label free quantification , proteomics , protein mass spectrometry , chromatography , peptide sequence , quantitative proteomics , enzyme , geotechnical engineering , fracture (geology) , engineering , gene
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.