Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini.

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Xu G, Shin SB, Jaffrey SR

Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini.

Proc Natl Acad Sci U S A. 2009 Nov 17;106(46):19310-5. doi: 10.1073/pnas.0908958106. Epub 2009 Nov 5.

PubMed ID
19892738 [ View in PubMed
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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.

DrugBank Data that Cites this Article

Polypeptides
NameUniProt ID
NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrialO75489Details
NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 2, mitochondrialO95178Details
Acyl carrier protein, mitochondrialO14561Details
Asparagine--tRNA ligase, cytoplasmicO43776Details
ATP-dependent Clp protease proteolytic subunit, mitochondrialQ16740Details
60S ribosomal protein L26-like 1Q9UNX3Details
ATP synthase subunit alpha, mitochondrialP25705Details
ATP synthase subunit beta, mitochondrialP06576Details
Cytochrome c oxidase subunit 6CP09669Details
1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenaseQ9BV57Details
ATP synthase subunit e, mitochondrialP56385Details