Analysis of disulfide-linked homo- and hetero-peptide dimers with a strong cation-exchange sulfoethyl aspartamide column.
A strong cation-exchange sulfoethyl aspartamide column was used to analyze and purify disulfide-linked homo-/hetero-peptide dimers. Monomeric peptides elute from this column in a monotonic fashion according to their net nominal positive charge at pH 3.0. Disulfide-linked peptide dimers are expected to possess an increased net positive charge at pH 3.0 and therefore should elute later and be well resolved from their monomeric constituents. Five distinct synthetic peptides (7 to 14 residues in length) ranging in net nominal charge at pH 3.0 from +1 to +4, with cysteine located at the N-terminus, C-terminus, or at an internal position, were used to produce disulfide-linked homo/hetero-peptide dimers as follows: The peptide was first reacted with 5,5'-dithiobis (2-nitrobenzoic acid) to form the mixed-disulfide, peptide:2-nitro-5-thiobenzoic acid adduct which is easily monitored at 325 nm. Then, the second cysteine-containing peptide was added and the desired disulfide-linked homo-/hetero-peptide dimer was produced via a thiol-disulfide interchange reaction. The entire reaction mixture was subsequently chromatographed on the sulfoethyl aspartamide column to isolate the disulfide-bonded species and also, to identify other reaction products. In addition, the same reaction mixture was analyzed by standard C18 reverse-phase chromatography to compare the capabilities of these two distinct chromatographic modes to identify disulfide-linked homo-/hetero- peptide dimers. It is shown that each chromatographic system successfully resolved all five homo-peptide dimers from their respective monomer counterparts, with separation being slightly better on the sulfoethyl aspartamide column.(ABSTRACT TRUNCATED AT 250 WORDS)
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Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110.
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003545:Cysteine; D004228:Dithionitrobenzoic Acid; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000032:analysis; D010455:Peptides / Q000032:analysis*; D013440:Sulfides
D007202:Indicators and Reagents; D046911:Macromolecular Substances; D009842:Oligopeptides; D010455:Peptides; D013440:Sulfides; D004228:Dithionitrobenzoic Acid; D003545:Cysteine