Electrospray ionization mass spectrometry of genetically and chemically modified bacteriorhodopsins

A simple method for the preparation of bacterio-rhodopsin for mass spectrometry is described, consisting of precipitation of the sample, removal of lipids by washing the sample, and dissolving it in an acidic mixture of organic solvents. Examples demonstrate the method's suitability for the analysis of genetically and chemically modified bacteriorhodopsins. The observed molecular masses are within 0.01% in accordance with the theoretical masses of the wild-type protein and modification products. This allows the detection of modifications accompanied by a mass change greater than 2 Da. Thus, only 9 of 380 possible binary amino acid exchanges are not amenable to analysis. Also chemical modifications of bacteriorhodopsin can be followed since it is possible to characterize reaction products and to approximate yields. However, secondary reactions like hydrolysis in the reaction medium or in the acid solvent used for sample preparation must be considered.