High-performance liquid chromatography of complex mixtures of cyanogen bromide-produced peptides from different proteins.
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.
Iadarola
P
P
Dipartimento di Biochimica, Università di Pavia, Italy.
Zapponi
M C
MC
Stoppini
M
M
Meloni
M L
ML
Minchiotti
L
L
Galliano
M
M
Ferri
G
G
D000418:Albumins / Q000032:analysis; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*
D000418:Albumins; D000596:Amino Acids; D007202:Indicators and Reagents; D010455:Peptides; D003488:Cyanogen Bromide