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.