Sequence Analysis of Membrane Protein Fragments Isolated by High Pressure Liquid Chromatography

Abstract

Sequence analysis of membrane proteins has been severely limited by the lack of effective chromatographic techniques for the separation of hydrophobic polypeptides. The crux of this problem is the insolubility of these peptides except under conditions which preclude the use of conventional chromatographic methods. Some progress has been made with gel permeation chromatography using organic solvents or concentrated organic acids, but these methods require large amounts of protein — something not usually available for membrane proteins. The recent advent of reverse phase high pressure liquid chromatography (RPHPLC) has revolutionized the field of peptide separation. Polar peptides in particular can be separated in good yield, with high resolution, and sensitivity in the picomole range by a variety of solvent systems. The application of RPHPLC to the isolation of hydrophobic peptides, however, is less well developed. We have previously shown that a 44 residue hydrophobic segment of cytochrome b 5 can be isolated by RPHPLC using dilute phosphoric acid and acetonitrile (1,2).