Synthesis and evaluation of a fluorogenic reagent for proteomic studies: 7-fluoro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-F).
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, beta-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.
Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi Tokyo, Japan 202-8585.
Akiyo
A
Koshiyama
Kazuhiro
K
Imai
D002853:Chromatography, Liquid; D004847:Epithelial Cells / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry*; D006801:Humans; D010069:Oxadiazoles / Q000138:chemical synthesis* / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics; D013449:Sulfonamides / Q000138:chemical synthesis* / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
C554066:7-fluoro-N-(2-(dimethylamino)ethyl)-2,1,3-benzoxadiazole-4-sulfonamide; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D011506:Proteins; D013449:Sulfonamides