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Synthesis and evaluation of fluorogenic reagents for simultaneous detection of peptides and proteins by HPLC in two different samples
Author(s) -
Saimaru Hiroshi,
Yasui Eiko,
Takamura Norio,
Imai Kazuhiro
Publication year - 2006
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.681
Subject(s) - reagent , chemistry , chromatography , fluorescence , high performance liquid chromatography , trypsin , excitation wavelength , organic chemistry , enzyme , physics , quantum mechanics
To find the pairs of fluorogenic reagents having similar retention times in HPLC but with different fluorescent characteristics, six fluorogenic reagents bearing benzoxadiazole or benzoselenadiazole skeletons were synthesized. The resultant derivatives obtained from the reaction of peptides and proteins with reagents which have a benzoselenadiazole skeleton showed different fluorescence characteristics from those with a benzoxadiazole skeleton. Since each corresponding derivatives of trypsin inhibitor and BSA with DAABD‐Cl and 7‐fluoro‐ N ‐[2‐(diethylamino)ethyl]‐2,1,3‐benzoselenadiazole‐4‐sulfonamide (DEAEABSeD‐F) have similar retention times, the pair of reagents was adopted for the sensitive simultaneous detection of proteins in two different samples. When the soluble fraction of mouse hippocampus was divided into the two samples (A and B), each was reacted with DEAEABSeD‐F for A and DAABD‐Cl for B, respectively. The two reaction solutions were combined and subjected to HPLC analysis with two fluorescent detectors in series (excitation and emission at different wavelengths for A and B, respectively). The resultant two chromatograms had quite similar patterns for each other. The new pair of fluorogenic reagents (DAABD‐Cl and DEAEABSeD‐F) would be applicable to proteomics studies using the previously reported FD‐LC‐MS/MS method. Copyright © 2006 John Wiley & Sons, Ltd.