262 amino acids (2, 3). The protein is processed at the A simple method for the preparation of bacterio-N-terminus in at least three steps by the removal of 7, rhodopsin for mass spectrometry is described, con-10, and 13 amino acids (4), and the final N-terminus sisting of precipitation of the sample, removal of lipforms a pyroglutamate residue (5). The C-terminus is ids by washing the sample, and dissolving it in an shortened by the last aspartic acid which gives the maacidic mixture of organic solvents. Examples demonture protein a length of 248 amino acids and an average strate the method's suitability for the analysis of gemolecular mass of 26,783.6 Da.
netically and chemically modified bacteriorhodop-Due to their insolubility, the chemistry and analysis sins. The observed molecular masses are within of membrane proteins are notoriously difficult. For the 0.01% in accordance with the theoretical masses of analysis of posttranslational processing and chemical the wild-type protein and modification products. modifications, as well as for the verification of muta-This allows the detection of modifications accompations introduced using the recently developed system nied by a mass change greater than 2 Da. Thus, only for site-directed mutagenesis and homologous expres-9 of 380 possible binary amino acid exchanges are not sion of halobacterial genes (6), mass spectrometry amenable to analysis. Also chemical modifications of would be a preferred technique if the problem of sample bacteriorhodopsin can be followed since it is possipreparation for membrane proteins could be solved. ble to characterize reaction products and to approxi-Two methods are frequently used for molecular mate yields. However, secondary reactions like hymass determination of proteins. These are matrixdrolysis in the reaction medium or in the acid solvent assisted laser desorption/ionization -mass spectromused for sample preparation must be considered.
etry (MALDI -MS, 7 -9) and electrospray ionization -