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Purification of hydrophilic and hydrophobic peptide fragments on a single reversed phase high performance liquid chromatographic column
Author(s) -
McKern Neil M.,
Edskes Herman K.,
Shukla Dharma D.
Publication year - 1993
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130070105
Subject(s) - chemistry , chromatography , peptide , elution , edman degradation , hydrophilic interaction chromatography , reversed phase chromatography , acetonitrile , high performance liquid chromatography , peptide sequence , biochemistry , gene
Hydrophilic peptides generated from enzymic fragmentation of proteins are difficult to purify because they are either weakly bound or unretained by the reversed phase C 18 columns favoured for liquid chromatographic separation of peptide mixtures. To overcome this difficulty, peptides that were not bound or only weakly bound by a C 18 RP column were reacted with phenyl isothiocyanate (PITC), as used in the initial step in Edman sequencing. The hydrophobic phenylthiocarbamyl (PTC) peptide derivatives produced by the reaction were rechromatographed on the same column. Peptides generated by tryptic digestion of equine cytochrome C were used as a model system to test whether a complete set of peptide fragments could be purified by this method using just one column and solvent system. All the expected hydrophobic tryptic peptides bound to the RP column and were resolved by elution with acetonitrile, but no hydrophilic peptides were recovered as pure fractions. The column breakthrough fraction was reacted with PITC and rechromatographed on the same column, producing a profile consisting of 19 bound peaks. Further rechromatography of some of the fractions at different column temperatures enabled all six of the expected hydrophilic peptides to be purified and identified. The technique has also been applied to the sequence determination of coat protein from peanut stripe potyvirus protein, eight hydrophilic tryptic peptides being recovered and identified as PTC derivatives.