| Title | Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini. | | Author(s) | Xu G, Shin SB, Jaffrey SR | | Institution | Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065. | | Source | Proc Natl Acad Sci U S A 2009 Nov 17; 106(46):19310-19315. | | Abstract | Proteolysis has major roles in diverse biologic processes and regulates the activity, localization, and intracellular levels of proteins. Linking signaling pathways and physiologic processes to specific proteolytic processing events is a major challenge in signal transduction research. Here, we describe N-CLAP (N-terminalomics by chemical labeling of the alpha-amine of proteins), a general approach for profiling protein N-termini and identifying protein cleavage sites during cellular signaling. In N-CLAP, simple and readily available reagents are used to selectively affinity label the alpha-amine that characterizes the protein N terminus over the more highly abundant epsilon-amine on lysine residues. Protein cleavage sites are deduced by identifying the corresponding N-CLAP peptides, which are derived from the N-termini of proteins, including the N-termini of the newly formed polypeptide products of proteolytic cleavage. Through selective affinity purification and tandem mass spectrometry analysis of 278 N-CLAP peptides, we characterized proteolytic cleavage events associated with methionine aminopeptidases and signal peptide peptidases, as well as proteins that are proteolytically cleaved after cisplatin-induced apoptosis. Many of the protein cleavage sites that are elicited during apoptotic signaling are consistent with caspase-dependent cleavage. These data demonstrate the utility of N-CLAP for proteomic profiling of protein cleavage sites that are generated during cellular signaling. | | Language | ENG | | Pub Type(s) | JOURNAL ARTICLE
| | PubMed ID | 19892738 |
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