Labeling analysis of three neuropeptides（Met-enkephalin, Leu-enkephalin, and neurotensin） with 7-methoxycoumarin-3-carboxylic acid N-succinimidyl ester（MCSE） was investigated. The experimental conditions for labeling reaction and HPLC analysis of the neuropeptide derivatives were optimized. It was found that the labeling reatction could be achieved at 37 ℃ for one hour in H3BO3-NaCl-Na2B4O7 buffer（pH=8.3） with a labeling ratio of about 80%. The derivative mixture from the three peptides can be separated in a 40 min linear gradient from 60% to 20% of solvent A（100 mmol/L sodium acetate, pH=5.0） and from 40% to 80% of solvent B[60%（volume fraction） acetonitrile in water]. The resulting labeled products were also confirmed by MALDI-TOF-MS analysis. Compared with the native neuropeptides, the resulting labeled products have a longer absorption and fluorescence wavelengths, which is beneficial to the effective elimination of background fluorescence interference from biological matrix.
A new and sensitive method for the analysis of amino acids or oligopeptides has been developed by using transamination for fluorescence derivatization. Namely, α-amino group of amino acid or N-terminal amino group of oligopeptide was selectively converted into a highly reactive carbonyl group by transamination, and then coupled with 2,3-diaminonaphthalene to yield a fluorescent compound (a benzoquinoxaline derivative). The product was detected by fluorescence spectoscopy and electrospray mass spectrometry.The product is a polynuclear aromatic compound with a higher fluorescence quantum yield. It has an excitation and emission wavelength of 364nm and 514nm, respectively.The MS analysis confirmed the formation of the expected benzoquinoxaline derivative with a molecular ion peak at m/z268. The whole operation was conducted under mild conditions, and the method may be used to the sensitive detection of amino acids or peptides. It should be noted that some difficulties were encountered in the separation of the reaction products and the control of the reaction conditions, which need to be further improved.
