title: A fluorescent derivatization method of proteins for the detection of low-level impurities by microchip capillary gel electrophoresis.
abstract: A novel pre-chip fluorescent derivatization method is presented for protein sizing and quantification by microchip CGE. The derivatization reaction employed a water-soluble and stable fluorescent dye and was performed under conditions that favored the formation of homogeneous reaction products. The method delivered in terms of protein sizing similar results as microchip CGE with on-chip staining but showed an extended linear dynamic range for protein quantification encompassing four orders of magnitude. The sensitivity of the method was similar to standard silver-stained planar gels. The characterization of derivatization reaction products by MS and preparative isoelectric focusing indicated that a constant degree of dye molecule tagging was obtained over a broad range of protein/dye ratios. The method allowed detecting and quantifying an impurity spiked into an antibody preparation down to a level of 0.05%. Advantages of this method compared with CGE approaches with pre-column derivatization include a shorter analysis time and an increased robustness and ease of use.
authors.affiliation: Agilent Technologies R&D and Marketing GmbH & Co KG, 76337Waldbronn, Germany. christian_wenz@agilent.com
authors.forename: Christian
authors.initials: C
authors.lastname: Wenz
authors.forename: Martina
authors.initials: M
authors.lastname: Marchetti-Deschmann
authors.forename: Ela
authors.initials: E
authors.lastname: Herwig
authors.forename: Evita
authors.initials: E
authors.lastname: Schröttner
authors.forename: Günter
authors.initials: G
authors.lastname: Allmaier
authors.forename: Lukas
authors.initials: L
authors.lastname: Trojer
authors.forename: Martin
authors.initials: M
authors.lastname: Vollmer
authors.forename: Andreas
authors.initials: A
authors.lastname: Rüfer
mesh_terms: D003198:Computer Simulation; D046209:Electrophoresis, Microchip / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007525:Isoelectric Focusing / Q000379:methods; D007526:Isoelectric Point; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins