title: Identification and relative quantification of native and proteolytically generated protein C-termini from complex proteomes: C-terminome analysis.
abstract: Proteome-wide analysis of protein C-termini has long been inaccessible, but is now enabled by a newly developed negative selection strategy we term C-terminomics. In this procedure, amine- and carboxyl groups of full-length proteins are chemically protected. After trypsin digestion, N-terminal and internal tryptic peptides - but not C-terminal peptides - posses newly formed, unprotected C-termini that are removed by coupling to the high-molecular-weight polymer poly-allylamine. Ultrafiltration separates the uncoupled, blocked C-terminal peptides that are subsequently analyzed by liquid chromatography-tandem mass spectrometry. On a proteome-wide scale, this strategy profiles native protein C-termini together with neo C-termini generated by endoproteolytic cleavage or processive C-terminal truncations ("ragging"). In bacterial proteomes, hundreds of protein C-termini were identified. Stable isotope labeling enables -quantitative comparison of protein C-termini and C-terminal processing in different samples. Using formaldehyde-based chemical labeling, this quantitative approach termed "carboxy-terminal amine-based isotope labeling of substrates (C-TAILS)" identified >100 cleavage sites of exogenously applied GluC protease in an Escherichia coli proteome. C-TAILS complements recently developed N-terminomic techniques for endoprotease substrate discovery and is essential for the characterization of carboxyprotease processing.
authors.affiliation: Institute for Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
authors.forename: Pitter F
authors.initials: PF
authors.lastname: Huesgen
authors.forename: Olivier
authors.initials: O
authors.lastname: Barré
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D002268:Carboxypeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism
chemical_list: D020543:Proteome; D002268:Carboxypeptidases