Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini.
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.
Xu
Guoqiang
G
Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065, USA.
Shin
Sung Bin Y
SB
Jaffrey
Samie R
SR
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D063208:Methionyl Aminopeptidases; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D010455:Peptides; D008715:Methionine; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase