Homogeneous fluorescent derivatization of large proteins

A method of homogeneously derivatizing large proteins for highly sensitive analysis is described. Homogeneity of the derivative was realized by tagging all the free amino groups of proteins. With this method, α-chymotrypsinogen A, ovalbumin and bovine serum albumin were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Prior to the derivatization, all the proteins were reduced and alkylated. After reacting the resulting unfolded proteins with excessive amounts of AQC, the samples were analyzed with matrix assisted laser desorption ionization–time of flight-mass spectrometry (MALDI–TOF-MS) to determine the derivatization degree. The results indicated that all three proteins had been, or had almost been, fully derivatized. HPLC and CE were used for characterizing these protein derivatives. Under 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.