Microderivatization of peptides by placing a fixed positive charge at the N-terminus to modify high energy collision fragmentation

Abstract

For the differentiation of leucine and isoleucine in a peptide by high energy collision-induced dissociation (CID), it is generally required that there is a basic amino acid present at or near either the C-terminus or the N-terminus of the peptide. In these cases, fragmentation or the β,γ bond of the side chain occurs, generating ions designated w n or d n that permit the differentiation of these isomeric amino acids. While trypsin and Endo Lys-C generate peptides with a basic C-terminal amino acid, other enzymes cleave at neutral or acidic amino acids and may thus produce proteolytic peptides that do not contain any basic amino acids. For these, a microderivatization method has been developed that places a fixed positive charge at the C-terminus. It involves exposure of the peptide(s) deposited on the inner wall of a capillary tube, first to chloroacetyl chloride vapor and then to trimethylamine and water vapors. This two-step reaction attaches a trimethylammonium acetyl (TMA) moiety to the N-terminal amino group of the peptide. The CID spectra of these derivatives are very simple, exhibit the same characteristics (including abundant d n ions) as peptides bearing the strongly basic arginine at the N-terminus, and thus permit the differentiation of leucine from isoleucine. The reaction can be carried out at the sub-nanomole level.