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Fluorescent derivatization method of proteins for characterization by capillary electrophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection.
Anal Chem. 2007 Aug 01; 79(15):5963-71.AC

Abstract

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.

Authors+Show Affiliations

Department of Process and Analytical Science, Amgen Incorporated, 1201 Amgen Court West, Seattle, Washington 98119, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

17591753

Citation

Michels, David A., et al. "Fluorescent Derivatization Method of Proteins for Characterization By Capillary Electrophoresis-sodium Dodecyl Sulfate With Laser-induced Fluorescence Detection." Analytical Chemistry, vol. 79, no. 15, 2007, pp. 5963-71.
Michels DA, Brady LJ, Guo A, et al. Fluorescent derivatization method of proteins for characterization by capillary electrophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection. Anal Chem. 2007;79(15):5963-71.
Michels, D. A., Brady, L. J., Guo, A., & Balland, A. (2007). Fluorescent derivatization method of proteins for characterization by capillary electrophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection. Analytical Chemistry, 79(15), 5963-71.
Michels DA, et al. Fluorescent Derivatization Method of Proteins for Characterization By Capillary Electrophoresis-sodium Dodecyl Sulfate With Laser-induced Fluorescence Detection. Anal Chem. 2007 Aug 1;79(15):5963-71. PubMed PMID: 17591753.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Fluorescent derivatization method of proteins for characterization by capillary electrophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection. AU - Michels,David A, AU - Brady,Lowell J, AU - Guo,Amy, AU - Balland,Alain, Y1 - 2007/06/26/ PY - 2007/6/27/pubmed PY - 2007/10/3/medline PY - 2007/6/27/entrez SP - 5963 EP - 71 JF - Analytical chemistry JO - Anal Chem VL - 79 IS - 15 N2 - 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. SN - 0003-2700 UR - https://www.unboundmedicine.com/medline/citation/17591753/Fluorescent_derivatization_method_of_proteins_for_characterization_by_capillary_electrophoresis_sodium_dodecyl_sulfate_with_laser_induced_fluorescence_detection_ L2 - https://doi.org/10.1021/ac0705521 DB - PRIME DP - Unbound Medicine ER -