Mass spectrometry is a standard method for the sequence determination of peptides, and is of particular use when dealing either with mixtures of peptides, or with post-translationally modified peptides. The MS methods used include (i) standard positive ion spectra (determined by FAB and other ionization techniques), (ii) linked scanning including MS/MS and MS/MS/MS, and (iii) manual Edman degradation using MS to determine the mass of the residual peptide. We have found it difficult to distinguish between isomeric Leu and Ile using these techniques. Biemannl has used side chain fragmentation to differentiate between these residues in some circumstances, but we have little success with this approach in our studies.
An alternative approach is to use manual/MS Edman degradation, but to apply MS to identify the phenylthiohydantoin amino acid derivative formed during the Edman process, rather than the mass of the residual peptide. This has been done previously using positive ion mass spectrometry.2
Our current interests include the study of negative ion mass spectrometry. Thus we have determined the HO- chemical ion negative ion mass spectra of all of the phenylthiohydantoin amino acids: they all show diagnostic spectra. In particular, Leu and Ile are readily differentiated by this technique. The technique has been tested on standard peptides: we can now routinely differentiate Leu and Ile residues with the manual Edman/MS technique using 10µg of material.