Analysis of cysteine-containing proteins using precolumn derivatization with N-(2-ferroceneethyl)maleimide and liquid chromatography/electrochemistry/mass spectrometry.
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.
Seiwert
Bettina
B
Chemical Analysis Group and MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500, AE Enschede, The Netherlands.
Karst
Uwe
U
D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002853:Chromatography, Liquid; D019411:Clinical Laboratory Techniques; D003545:Cysteine / Q000032:analysis / Q000737:chemistry*; D004563:Electrochemistry; D005296:Ferrous Compounds / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry*; D013058:Mass Spectrometry; D000075163:Metallocenes; D011506:Proteins / Q000032:analysis*
D005296:Ferrous Compounds; D008301:Maleimides; D000075163:Metallocenes; C100759:N-(2-ferrocene-ethyl)maleimide; D011506:Proteins; D003545:Cysteine; C004998:ferrocene