Protocol

Desalting of Peptides and Protein Mixtures by RP-HPLC Techniques

This protocol was adapted from "Reversed-Phase High-Performance Liquid Chromatography," Chapter 5 in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

The RP-HPLC technique can be used to "desalt" peptide or protein samples derived from extraction procedures, from chemical reactions such as reductive alkylation in the presence of urea or guanidine hydrochloride, citraconylation, iodination, or cyanogen bromide cleavage, or recovered from other chromatographic separation. The peptide or protein solution is injected onto a small RP-HPLC column. An aqueous buffer is used to elute the salts while the peptides or proteins are concentrated at the top of the column. After elution of the salts, monitored by UV detection, the peptides or proteins are eluted with H₂O-acetonitrile or H₂O-isopropanol mobile phases.