Applying Arginylation for Bottom-Up Proteomics

Abstract

Arginylation is an enzymatic reaction in which arginyl-tRNA protein transferase 1 (ATE1, EC 2.3.2.8) conjugates a single arginyl moiety from aminoacylated tRNA Arg onto a target polypeptide. We established arginylation for in vitro labeling of peptides with N-terminal acidic amino acids. Consistent with prior knowledge, arginylated peptides fl anked by basic amino acids result in rich redundant MS/MS fragment spectra using various precursor fragmentation modes. Arginylation carried out by ATE1 is a fast method for labeling peptides. Sequence-specifi c proteolytic digest of proteins is best carried out using a double digest of proteins by Lys-C and Asp-N to generate peptides with a basic amino acid on the C-terminus and an acidic amino acid on the N-terminus. Under these conditions, arginylation is specifi c for N-terminal acidic amino acids and results in a near 2× sequence coverage in the MS/MS spectrum are achieved.