IDENTIFICATION OF CYSTEINE-CONTAINING PEPTIDES DURING THE PEPTIDE-MAPPING OF RECOMBINANT PROTEINS

Confirmation of a protein's cysteine content and of its location withi n the amino acid sequence is crucial in investigating the structural i ntegrity of recombinant proteins. A combination of thiol-specific chem istry 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 tum or necrosis factor alpha. Recombinant proteins were conjugated with a hydrophobic iodoacetamide derivative, dimethylaminoazo-benzene iodoace tamide, and digested with trypsin. The peptide fragments were separate d on a C-8 microbore reversed-phase column using a linear acetonitrile gradient. The peptides containing the cysteine residues were selectiv ely identified by monitoring with a diode-array detector at 215 nm wit h the reference wavelength set at 450 nm. Cysteine-containing peptides could be readily distinguished as inverted ''negative'' peaks relativ e to the baseline and noncysteine-containing peptides. Isolated peptid e fragments were then sequenced in order to confirm the location of th e cysteines in the proteins. This approach offers the benefits of sele ctively 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 hydrop hobic, thereby making them easier to separate from noncysteine-contain ing peptides.