Differential labeling of free and disulfide-bound thiol functions in proteins.
A method for the simultaneous determination of the number of free cysteine groups and disulfide-bound cysteine groups in proteins has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. Liquid chromatography/electrochemistry/mass spectrometry was used to assign the N-(2-ferroceneethyl)maleimide (FEM) labeled free cysteine functionalities in a tryptic digest mixture, whereas a precursor ion scan enables the detection of peptides with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA) labeled disulfide-bound cysteine groups after reduction. Fragment spectra of the labeled peptides yield an excellent coverage of b-type and y-type ions. The ferrocene labeled cysteines were fragmented as 412 Da (FEM) and 455 Da (FMEA). These fragment masses are significantly higher than unlabeled amino acids or dipeptides and are easily detected. The position of free and disulfide-bound cysteine may therefore be assigned in an amino acid sequence.
Seiwert
Bettina
B
Chemical Analysis Group and MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands.
Hayen
Heiko
H
Karst
Uwe
U
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D019411:Clinical Laboratory Techniques; D003545:Cysteine / Q000032:analysis / Q000737:chemistry*; D004220:Disulfides / Q000032:analysis / Q000737:chemistry*; D004563:Electrochemistry; D005296:Ferrous Compounds / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry*; D000075163:Metallocenes; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D004220:Disulfides; D005296:Ferrous Compounds; D008301:Maleimides; D000075163:Metallocenes; C100759:N-(2-ferrocene-ethyl)maleimide; D011506:Proteins; D003545:Cysteine