A new labeling method compatible with both laser-induced fluorescence (LIF) and MS detection for enkephalins, which uses naphthalene-2,3-dicarboxaldehyde (NDA) and a new nucleophilic agent (N,N-dimethylaminoethanethiol) is described. When the derivative is separated via reverse phase HPLC and detected via MS, two different peaks with similar exact mass but different fluorescence and fragmentation properties are obtained. To interpret these results, molecular modeling and H/D exchange mass spectrometry studies were investigated to test the hypothesis that the peak obtained by LC/LIF/MS analysis depends on the site of protonation of the labeled enkephalins. The peptides labeled with NDA and N,N-dimethylaminoethanethiol were separated on a reverse phase C18 column with a gradient of aqueous 0.1% formic acid and acetonitrile. In mass spectrometry, two peaks are observed with the same exact mass for each molecule while only one peak is detected using fluorescence. Tandem mass spectrometry experiments of ion m/z 809.5 were performed on each chromatographic peak; the first peak (which is not observed by LIF detection) gives a fragment corresponding to the loss of the aminothiol side chain while no fragmentation is observed on the second peak, which was detected by fluorescence. The hypothesis is that each peak represents the labeled enkephalin with different sites of protonation. According to this hypothesis, three fundamental conformations that were closed to the unlabeled leucine-enkephalin were obtained by molecular modeling: a beta-turn like conformation with two hydrogen bonds, a 3(10)-helix with an H bond, and finally, the extended form without any intramolecular interactions. H/D exchange mass spectrometry experiments with D(2)O and d(2-)formic acid as eluent was used to determine which conformation is involved in each peak.