A new fluorogenic reagent aimed at simultaneous determination of peptides or proteins in two different samples

Abstract

Previously, we have synthesized a thiol specific fluorogenic reagent, 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) and derivatized protein mixtures, isolated the derivatives by HPLC, followed by enzymatic digestion and identification of the derivatized proteins by HPLC-electrospray ionization mass spectrometry (ESI-MS/MS) with the probability-based protein identification algorithm (FD-LC-MS/MS method) (M. Masuda et al., Anal. Chem., 2004, 76, 728). The emission wavelengths for the DAABD derivatives of proteins were 510 nm with an excitation wavelength of 380 nm. In this paper, a new thiol specific fluorogenic reagent, 7-fluoro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoselenadiazole-4-sulfonamide (DAABSeD-F) was synthesized. It reacted with thiols of peptides and proteins to give fluorescence. The respective excitation and emission wavelengths of the DAABSeD derivatives of proteins were about 30 nm and 40 nm red shifted from those of the DAABD proteins. These properties would be appropriate for its use in combination with DAABD-Cl as a counterpart for the simultaneous detection, ratio calculation and identification of peptides or proteins produced under the different conditions. Thus, DAABSeD-F could be an additional supportive reagent for the FD-LC-MS/MS method.