期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2009
卷号：106
期号：46
页码：19310-19315
DOI：10.1073/pnas.0908958106
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 {varepsilon}-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.
关键词：caspase ; N-CLAP ; N-terminalomics ; tandem mass spectrometry
