Fluorescent derivatization method of proteins for characterization by capillary electrophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection.
A fast and improved sample preparation scheme was developed for protein analysis using capillary electrophoresis-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 degrees 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.
Michels
David A
DA
Department of Process and Analytical Science, Amgen Incorporated, 1201 Amgen Court West, Seattle, Washington 98119, USA.
Brady
Lowell J
LJ
Guo
Amy
A
Balland
Alain
A
D000478:Alkylation; D000911:Antibodies, Monoclonal / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005663:Furans / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D011506:Proteins / Q000032:analysis*; D011804:Quinolines / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D013997:Time Factors
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D005663:Furans; D011506:Proteins; D011804:Quinolines; D011994:Recombinant Proteins; C063756:3-(2-furoyl)quinoline-2-carbaldehyde; D012967:Sodium Dodecyl Sulfate