High-performance liquid chromatography of complex mixtures of cyanogen bromide-produced peptides from different...

A variety of approaches have been required in order to achieve the resolution of large fragments from cyanogen bromide (CNBr) digests of Inc k chain (an immunoglobulin light chain), human serum albumin (HSA) and four of its mutants. Reversed-phase high-performance liquid chromatography (RP-HPLC) under different conditions failed to resolve the Inc k chain digest; the three CNBr fragments (3.1, 6.7 and 13.7 kDa) were separated in a homogeneous form by gel HPLC. Five of the seven CNBr fragments (ranging from 3.4 to 20.0 kDa) obtained from CNBr cleavage of HSA can be resolved by a single reversed-phase HPLC step; separation of the other two requires modification of the eluent composition. Some structural features of the peptides seem to influence their chromatographic behaviour; by examining the elution patterns from albumin mutants, the sequence-related contribution of single amino acid residues is apparent.