Fluorescent Derivatization Method of Proteins for Characterization by Capillary Electrophoresis-Sodium Dodecyl Sulfate with Laser-Induced Fluorescence Detection

摘要

A fast and improved sample preparation scheme wasdeveloped for protein analysis using capillary electro-phoresis-sodium dodecyl sulfate (CE-SDS) with laser-induced fluorescence detection. This CE-SDS method was developed as a purity assay for recombinant monoclonal antibodies (rMAbs). In this assay, rMAbs are derivatized with the fluorogenic reagent 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ) in the presence of a nucleophile (CN~(-)), which fluoresces only upon covalent binding to the protein. Purification after labeling is therefore not necessary to remove unreacted reagents. Proteins are incubated at 75 deg C for 5 min to facilitate denaturation and labeling. For nonreduced preparation, rMAbs are labeled at pH 6.5 with a dye-to-protein (D/P) molar ratio of 50:1, which forms conjugates having 6 +- 4 FQ labels. For reduced preparation, rMAbs are labeled at pH 9.3 with a D/P molar ratio of 10:1, which generates light chain conjugates incorporated with 3 +- 2 FQ labels. Labeling artifacts such as fragmentation or aggregation are absent with use of alkylation reagents. This efficient labeling scheme generates detection limits for FQ-labeled rMAbs as low as 10 ng/mL. In comparison to other labeling strategies, labeling proteins with FQ has the advantage of speed, ease of use, and robust quantification.