Advances in gas chromatographic mass spectrometric protein sequencing. 2--Application to membrane proteins.

The primary structure of the integral membrane protein bacteriorhodopsin was determined by an efficient combination of gas chromatographic mass spectrometric techniques with the Edman degradation. This combination of methodologies circumvented many of the experimental difficulties associated with the insolubility of bacteriorhodopsin and its primary degradation fragments in aqueous buffers. Specifically, in the gas chromatographic mass spectrometric analysis of the cyanogen bromide peptides derived from bacteriorhodopsin, it has been possible to identify homoserine-containing peptides which served as a starting point for the construction of C-terminal sequences. In most cases this C-terminal sequence constructed from the gas chromatographic mass spectrometric peptides overlapped the N-terminal sequence derived in an Edman degradation experiment, thereby completing the structure of the fragment. Furthermore, the specific identification of methionine-containing peptides required to establish the order of the cyanogen bromide fragments was accomplished by direct analysis of the complex mixtures generated by partial hydrolysis of segments of the protein. These data made it possible to determine the sequence of a large portion of bacteriorhodopsin solely from cyanogen bromide cleavage, one of the few specific reactions compatible with the solubility properties of this hydrophobic protein. Finally, the gas chromatographic mass spectrometric sequence data have been used to assign or confirm amino acids where the Edman data was ambiguous. These gas chromatographic mass spectrometric techniques resulted in an efficient and reliable determination of the complete sequence of this membrane protein which is 248 amino acids long.