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

Name	UniProt ID
NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial	O75489	Details
NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 2, mitochondrial	O95178	Details
Acyl carrier protein, mitochondrial	O14561	Details
Asparagine--tRNA ligase, cytoplasmic	O43776	Details
ATP-dependent Clp protease proteolytic subunit, mitochondrial	Q16740	Details
60S ribosomal protein L26-like 1	Q9UNX3	Details
ATP synthase subunit alpha, mitochondrial	P25705	Details
ATP synthase subunit beta, mitochondrial	P06576	Details
Cytochrome c oxidase subunit 6C	P09669	Details
1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase	Q9BV57	Details
ATP synthase subunit e, mitochondrial	P56385	Details