Structural elucidation of dipeptides displaying limited mass spectral information by liquid chromatography-electrospray ionization-tandem mass spectrometry

Small peptides, such as dipeptides, have attracted attention in many research fields because of their important biological functions and potential roles as disease biomarkers. However, the identification of many of them by implementation of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is a challenging task. This is because many dipeptides display limited mass spectral information in LC-ESI-MS/MS analyses, which leads to unavoidable ambiguity in determinations of their structures. In this study, two useful analytical techniques were developed for the structural elucidation of 10 representative dipeptides exhibiting a dominant/single product ion in the ESI-MS/MS spectra of the protonated molecules [M + H](+). Structural elucidation was obtained instantaneously through LC-ESI-MS/MS fragmentation of the accompanying sodium adducts [M + Na](+) or alternatively by "in-vial" chemical derivatization with isobutyl chloroformate. The sodium adducts and the resulting carbamate derivatives altered the charge distribution occurring during ESI-MS/MS fragmentation, enabling detailed structural elucidation and unambiguous identification of such dipeptides at ng/ml levels. These quick, simple, and easy techniques can be implemented to identify various dipeptides or confirm their identities without the need for complex sample handling.