N-CLAP: global profiling of N-termini by chemoselective labeling of the alpha-amine of proteins.

ABSTRACT: N-terminalomics identifies proteins by selectively enriching for and sequencing their N-terminal peptides using mass spectrometry (MS) in a high-throughput manner. Several N-terminalomics procedures have been developed to identify protein cleavage sites in a variety of cell-signaling events, such as apoptosis and N-terminal methionine excision. This protocol describes a newly developed N-terminalomics approach, N-CLAP (N-terminalomics by chemical labeling of the ?-amine of proteins). N-CLAP uses Edman chemistry to modify all of the amines in proteins, followed by the generation of a new unmodified amine at the N terminus after the removal of the first amino acid by peptide bond cleavage. A commercially available N-hydroxysuccinimide reagent is used to label the ?-amine at the protein N terminus with a cleavable biotin affinity tag, which facilitates the downstream purification of the N-terminal peptides. Peptides are eluted by cleaving the biotin affinity tag using reducing agent and identified by tandem mass spectrometry (MS/MS). N-CLAP can be used for the identification of signaling peptides for mature proteins as well as for global profiling of cleavage events that occur during cell signaling, such as apoptosis.

SUBMITTER: Xu G 

PROVIDER: S-EPMC3205986 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature