Synthesis and evaluation of a fluorogenic reagent for proteomic studies: 7-fluoro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-F)

Abstract

Since the successful selection of fluorogenic derivatization reagent 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as a component of a novel method (FD-LC-MS/MS method) for proteomics studies, a further reactive reagent has been required to obtain more species of proteins: DAABD-Cl reacts with only thiol moieties of proteins to give fluorescence at 505 nm with excitation at 395 nm. Here, we synthesized reagent 7-fluoro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide, DAABD-F, having a 7-fluorine moiety instead of the 7-chlorine moiety in DAABD-Cl, expecting it to exhibit high reactivity to amino moieties of proteins. As expected, the reaction rates of low molecular thiols with DAABD-F were 50 times higher than those with DAABD-Cl. DAABD-F was able to react with the amino moiety of a low molecular amine, β-alanine, producing fluorescence at 554 nm with excitation at 432 nm. The reaction with DAABD-F of a typical model protein, bovine serum albumin (BSA), needed a lower amount of reagent (DAABD-F) than DAABD-Cl to produce a single fluorescent derivative (fluorescence at 495 nm with excitation at 390 nm) that was demonstrated to be solely a cysteinyl residue modified product. A derivatization reaction with DAABD-F towards a soluble extract of a normal human mammary epithelial cell (HMEC) resulted in the same fluorescent protein profiles as those with DAABD-Cl except one (AHNAK nucleoprotein isoform1) that was produced by the derivatization at a lysinyl residue (4761Lys) and was identified according to the usual procedure of isolation and tryptic digestion of the fluorescent protein peak on the chromatogram and final LC-MS/MS with a database-searching algorithm.