MICRODERIVATIZATION OF PEPTIDES BY PLACING A FIXED POSITIVE CHARGE AT THE N-TERMINUS TO MODIFY HIGH-ENERGY COLLISION FRAGMENTATION

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 wn or dn 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 dn 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. © 1990.