Identification of cysteine-containing peptides during the peptide mapping of recombinant proteins.
Confirmation of a protein's cysteine content and of its location within the amino acid sequence is crucial in investigating the structural integrity of recombinant proteins. A combination of thiol-specific chemistry and peptide mapping by reversed-phase microbore HPLC was used to confirm the presence and map the location of cysteine residues in the primary sequences of recombinant porcine growth hormone and human tumor necrosis factor alpha. Recombinant proteins were conjugated with a hydrophobic iodoacetamide derivative, dimethylaminoazo-benzene iodoacetamide, and digested with trypsin. The peptide fragments were separated on a C8 microbore reversed-phase column using a linear acetonitrile gradient. The peptides containing the cysteine residues were selectively identified by monitoring with a diode-array detector at 215 nm with the reference wavelength set at 450 nm. Cysteine-containing peptides could be readily distinguished as inverted "negative" peaks relative to the baseline and noncysteine-containing peptides. Isolated peptide fragments were then sequenced in order to confirm the location of the cysteines in the proteins. This approach offers the benefits of selectively and rapidly identifying, from a single chromatrophic step, the cysteine-containing peptides of proteins. Furthermore, the use of the labeling reagent renders the cysteine-containing peptides more hydrophobic, thereby making them easier to separate from noncysteine-containing peptides.
Cardamone
M
M
Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne.
Ashman
K
K
Brandon
M R
MR
Puri
N K
NK
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis*; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis*; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry*; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D007202:Indicators and Reagents; D010446:Peptide Fragments; D011994:Recombinant Proteins; C038560:4-dimethylaminoazobenzene-4'-iodoacetamide; D004124:p-Dimethylaminoazobenzene; D003545:Cysteine