A new method for the selective isolation of cysteine-containing peptides. Specific labelling of the thiol group with a hydrophobic chromophore.
A new method for the selective isolation of cysteine-containing peptides was designed. The method is based on the specific labelling of thiol groups with a hydrophobic chromophore followed by enzymic fragmentation of the labelled protein and reversed-phase high-pressure liquid-chromatographic separation of the peptide mixture. This new method has several distinct advantages: (1) the hydrophobic-chromophore-labelled cysteine-containing peptides are easily separated from non-cysteine-containing peptides by reversed-phase high-pressure liquid chromatography; (2) only cysteine-containing peptides are detected in the visible region with sensitivity at the low picomole level; this high sensitivity allows isolation of nanogram amounts of pure cysteine-containing peptide; (3) during sequence determination of the chromophore-labelled cysteine-containing peptides, the cysteine residues are released as coloured anilinothiazolinone derivatives and can be detected directly in the picomole range; (4) with proteins bearing several disulphide groups, each disulphide group may undergo a different degree of reduction, and therefore the recovery of individual cysteine-containing peptides may be used to deduce the disulphide linkages present in the native protein. Two thiol-specific reagents, 4-dimethylaminoazobenzene-4'-iodoacetamide and 4-dimethylaminoazobenzene-4'-N-maleimide, were synthesized and characterized. The method was successfully used to isolate five cysteine-containing peptides from a completely reduced monoclonal-antibody kappa-light chain raised against the azobenzenearsonate determinant and six cysteine-containing peptides from a kappa-light chain raised against streptococcal group A polysaccharide. The principle of this method is applicable to the isolation of any peptide containing amino acid residues that can be specifically labelled with a hydrophobic chromophore.
D000485:Allergens; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis*; D007147:Immunoglobulin Light Chains; D007202:Indicators and Reagents; D010446:Peptide Fragments / Q000032:analysis / Q000302:isolation & purification*; D010940:Plant Proteins; D011058:Pollen; D013438:Sulfhydryl Compounds; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives / Q000138:chemical synthesis
C038561:4-dimethylaminoazobenzene-4'-N-maleimide; D000485:Allergens; C026967:Amb a I protein, Ambrosia artemisiifolia; D052179:Antigens, Plant; D007147:Immunoglobulin Light Chains; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010940:Plant Proteins; D013438:Sulfhydryl Compounds; C038560:4-dimethylaminoazobenzene-4'-iodoacetamide; D004124:p-Dimethylaminoazobenzene; D003545:Cysteine