Homogeneous fluorescent derivatization of large proteins

Citation
Hj. Liu et al., Homogeneous fluorescent derivatization of large proteins, J CHROMAT A, 927(1-2), 2001, pp. 77-89
Citations number
37
Categorie Soggetti
Chemistry & Analysis","Spectroscopy /Instrumentation/Analytical Sciences
Journal title
Volume
927
Issue
1-2
Year of publication
2001
Pages
77 - 89
Database
ISI
SICI code
Abstract
A method of homogeneously derivatizing large proteins for highly sensitive analysis is described. Homogeneity of the derivative was realized by taggin g all the free amino groups of proteins. With this method, a-chymotrypsinog en A, ovalbumin and bovine serum albumin were derivatized with 6-aminoquino lyl-N-hydroxysuccinimidyl carbamate (AQC). Prior to the derivatization, all the proteins were reduced and alkylated. After reacting the resulting unfo lded proteins with excessive amounts of AQC, the samples were analyzed with matrix assisted laser desorption ionization-time of flight-mass spectromet ry (MALDI-TOF-MS) to determine the derivatization degree. The results indic ated that all three proteins had been, or had almost been, fully derivatize d. HPLC and CE were used for characterizing these protein derivatives. Unde r the optimized fluorescence detection conditions, the detectability of the tagged proteins was 2400-6200 times better than that detected at UV 280 nm , 170-300 times better than detected at UV 214 nm, and 150-420 times better than measured with their native fluorescence. (C) 2001 Elsevier Science B. V. All rights reserved.