Hydrophilic interaction/cation-exchange chromatography for the purification of synthetic peptides from closely related impurities: serine side-chain acetylated peptides

• Published in: The journal of peptide research Vol. 54; no. 1; pp. 1 - 11
• Main Authors: Litowski, J.R., Semchuk, P.D., Mant, C.T., Hodges, R.S.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Munksgaard International Publishers 01.07.1999; Wiley
• Subjects: Acetylation; Amino Acid Sequence; Biochemical Research Methods; Biochemistry & Molecular Biology; cation-exchange chromatography; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange - methods; hydrophilic interaction chromatography; Life Sciences & Biomedicine; Mass Spectrometry; Molecular Sequence Data; Peptides - chemistry; Peptides - isolation & purification; Science & Technology; Serine - chemistry; serine side-chain acetylation; synthetic peptide purification; hydrophilic interaction chromatography; PERFORMANCE LIQUID-CHROMATOGRAPHY; serine side-chain acetylation; synthetic peptide purification; cation-exchange chromatography; RETENTION; ION; SEPARATION; HISTONES
• ISSN: 1397-002X; 1399-3011
• DOI: 10.1034/j.1399-3011.1999.00066.x

: Mixed‐mode hydrophilic interaction/cation‐exchange chromatography (HILIC/CEC) is a novel HPLC technique which has excellent potential for peptide separations. Separations by HILIC/CEC are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlayed on ionic interactions with the cation‐exchange matrix. Complex peptide mixtures produced by solid‐phase synthesis are a frequently encountered and challenging purification problem. In the present study a two‐step protocol, consisting of HILIC/CEC followed by RPC, was required for the successful purification of a 21‐residue synthetic amphipathic α‐helical peptide from serine side‐chain acetylated impurities, with HILIC/CEC proving to be highly sensitive to subtle differences in hydrophilicities between the acetylated peptides and the desired product. Investigation of the three potential sites of serine acetylation through solid‐phase synthesis of acetylated analogues of the desired peptide (peptides of the same sequence and secondary structure, but acetylated at different positions on the hydrophilic face of the α‐helix) demonstrated that acetylation was occurring at different sites on the peptide. HILIC/CEC was able to take advantage of very subtle changes in environment around the acetylation sites and thus effect a separation of these analogues not achievable by RPC or CEC alone.