Analysis of cysteine-containing proteins using precolumn derivatization with N-(2-ferroceneethyl)maleimide and liquid chromatography/electrochemistry/mass spectrometry. 2007

Bettina Seiwert, and Uwe Karst
Chemical Analysis Group and MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500, AE Enschede, The Netherlands.

N-(2-ferroceneethyl)maleimide (FEM) is introduced as an electroactive derivatizing agent for thiol functionalities in proteins. Using appropriate reaction conditions, the derivatization is completed within five minutes and no unspecific labeling of free amino functions is observed. Liquid chromatography/electrochemistry/mass spectrometry was used to detect the reaction products. The reagent is a useful tool for determining the number of free thiol groups or the total number of free and disulfide-bound thiol groups in proteins. The electrochemical cell provides additional information, because the increase in mass spectrometric response upon electrochemical oxidation of the neutral ferrocene to the charged ferrocinium groups is monitored. The method was successfully applied to the analysis of native proteins and their tryptic digests.

UI MeSH Term Description Entries
D008301 Maleimides Derivatives of maleimide (the structural formula H2C2(CO)2NH) containing a pyrroledione ring where the hydrogen atom of the NH group is replaced with aliphatic or aromatic groups.
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D002623 Chemistry Techniques, Analytical Methodologies used for the isolation, identification, detection, and quantitation of chemical substances. Analytical Chemistry Techniques,Analytical Chemistry Methods,Analytical Chemistry Method,Analytical Chemistry Technique,Chemistry Method, Analytical,Chemistry Methods, Analytical,Chemistry Technique, Analytical,Method, Analytical Chemistry,Methods, Analytical Chemistry,Technique, Analytical Chemistry,Techniques, Analytical Chemistry
D002853 Chromatography, Liquid Chromatographic techniques in which the mobile phase is a liquid. Liquid Chromatography
D003545 Cysteine A thiol-containing non-essential amino acid that is oxidized to form CYSTINE. Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D004563 Electrochemistry The study of chemical changes resulting from electrical action and electrical activity resulting from chemical changes. Electrochemistries
D005296 Ferrous Compounds Inorganic or organic compounds that contain divalent iron. Compounds, Ferrous
D000075163 Metallocenes Organometallic compounds that generally consist of two cylcopentadiene ANIONS joined in their centers by a metallic cation such as NICKEL; IRON; or TITANIUM. Ferrocenes
D013058 Mass Spectrometry An analytical method used in determining the identity of a chemical based on its mass using mass analyzers/mass spectrometers. Mass Spectroscopy,Spectrometry, Mass,Spectroscopy, Mass,Spectrum Analysis, Mass,Analysis, Mass Spectrum,Mass Spectrum Analysis,Analyses, Mass Spectrum,Mass Spectrum Analyses,Spectrum Analyses, Mass
D019411 Clinical Laboratory Techniques Techniques used to carry out clinical investigative procedures in the diagnosis and therapy of disease. Clinical Laboratory Test,Clinical Laboratory Testing,Clinical Laboratory Diagnoses,Clinical Laboratory Testings,Clinical Laboratory Tests,Diagnoses and Laboratory Examinations,Diagnosis, Laboratory,Laboratory Diagnosis,Laboratory Examinations and Diagnoses,Laboratory Techniques, Clinical,Clinical Laboratory Technique,Diagnose, Clinical Laboratory,Laboratory Diagnoses,Laboratory Technique, Clinical,Laboratory Test, Clinical,Laboratory Testing, Clinical,Technique, Clinical Laboratory,Test, Clinical Laboratory,Testing, Clinical Laboratory

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