Analysis of Hydrophobic Proteins and Peptides by Electrospray Ionization Mass Spectrometry

Abstract

During the last decade mass spectrometry has become an essential tool for the analysis of peptides and proteins. Electrospray ionization mass spectrometry (ESIMS) is one of several recently developed techniques for the determination of accurate molecular masses of proteins, peptides, and other biopolymers up to >100 kDa. Up to the present, analyses have been performed mainly on biopolymers that are soluble in aqueous solutions. Mass spectrometric analyses of very hydrophobic species, such as membrane proteins, have seldom been reported in the literature. This is mainly due to the incompatibility between most mass spectrometric techniques and detergents and/or salts which are required to retain such proteins in solution. Hydrophobic proteins (for example, bacterioopsin) and pep-tides are in general not soluble in the solutions (methanol/water or acetonitrile/water) typically used for ESIMS, and most detergents and chaotropes interfere with the analysis. We have developed sample handling protocols and solvent systems that are compatible with instrumental requirements and also are capable of retaining very hydrophobic peptides and proteins in solution. Chloroform/methanol/water mixtures were found to work well with, e.g., bacterioopsin, and also to be compatible with samples dissolved in hexafluoroisopropanol and 70-95% formic acid.