Ion-exchange high-performance liquid-chromatography steps in peptide purifications.
Ion-exchange high-performance liquid chromatography (HPLC; on Ultropac TSK DEAE and CM) is compared with conventional soft-gel ion-exchange chromatography in identical peptide purifications. The results show that separating properties are similar, but as expected, ion-exchange HPLC has a much higher resolving capacity and a higher sensitivity, and allows a considerably shorter total separation time. The same buffer systems as for conventional ion-exchange chromatography can be used, including urea to solubilize large peptides, if care is taken not to exceed the pH limits set by the column matrix. A complete purification scheme by HPLC in the nanomolar range, utilizing exclusion, ion-exchange, and reverse-phase chromatographies, is given with a complex peptide mixture from a digest of a large protein. Similar steps as in conventional soft-gel schemes can be utilized. It is concluded that ion-exchange HPLC is a suitable complement to commonly used reverse-phase HPLC steps and that it permits high speed and sensitivity over wide ranges of peptide sizes and amounts.
D000444:Aldehyde Dehydrogenase; D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D006736:Horses; D008099:Liver / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000302:isolation & purification*
D000596:Amino Acids; D010446:Peptide Fragments; D010455:Peptides; D000445:Aldehyde Oxidoreductases; D000444:Aldehyde Dehydrogenase