Separation of the Sticky Peptides from Membrane Proteins by High-performance Liquid Chromatography in a Normal-phase System

The amphiphilic peptides obtained upon cleavage of membrane proteins, including numerous receptors, are recalcitrant to most separation techniques as a consequence of their limited solubility and tendency to aggregate and adsorb to surfaces. This paper describes HPLC systems that can separate these "sticky" peptides on silica and aminopropyl-modified silica columns with a mobile phase consisting of a mixture of chloroform/methanol/isopropylamine. The protocols developed have been applied to synthetic M1 and M2 peptides, which constitute part of the transmembrane domain of glutamate-gated ion-channel proteins. Four of these M1 and M2 peptides were separated from minor synthetic impurities, and a 23-mer was baseline separated from a 28-mer. The HPLC procedures have also led to purification of the 10 peptides resulting from cyanogen bromide cleavage of bacteriorhodopsin, peptides which have so far eluded HPLC separation despite numerous attempts. These HPLC protocols have been used to purify peptides ranging from 4 to 50 amino acids in high yield while the columns continued to resolve sharp peaks after more than 100 separation runs over a 6-month period. These new HPLC systems offer an efficient method for the isolation and analysis of this important albeit troublesome class of peptides.