N-CLAP: Global Profiling of N-Termini by Chemoselective Labeling of the α-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 α-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 α-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.