Reverse-Phase HPLC Purification of Peptides from Natural Sources for Structural Analysis

Abstract

The continued modifications in peptide/protein sequencer hardware, derivatization and coupling chemistry, reagent delivery, and component detection provide the protein/peptide chemist with the tools to determine primary structural information on sub-picomole quantities of material. This dramatic quantum leap in sequencer sensitivity has made the preparation of samples even more critical with respect to purity. The task of isolating several hundred femtomoles or a few picomoles of a peptide of interest from a crude extract of cells or tissues seems daunting at first but is readily achievable if several important criteria are met and the requisite chromatographic hardware is available. Much of what is described for the fractionation of crude extracts of tissues or cells by reverse-phase high-performance liquid chromatography (HPLC) for the purposes of peptide isolation is equally applicable to the fractionation of protein digests for peptide-mapping purposes.