ANALYSIS OF HYDROPHOBIC PROTEINS AND PEPTIDES BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

Bacterioopsin (BO) from Halobacterium halobium, as well as its V8-prot ease and CNBr fragments from the C-terminal region, were used to estab lish appropriate conditions for the mass determination of membrane pro teins and peptides by electrospray mass spectrometry (ESI-MS). Of the tested solvents neat formic acid gave the best results for BO (26 781. 8 +/- 5.1 Da) and chloroform + methanol + water (2:5:2, v/v/v/) contai ning 2% acetic acid was best for the BO fragments (2461.5 +/- 0.2 Da, 7147.1 +/- 0.5 Da, 7748.0 +/- 0.4 Da) with an overall mass accuracy of about 0.01%. The three subunits IIAB(Man), IICMan and IIDMan of the m annose transporter complex in E. coli were used to establish condition s for liquid chromatography/electrospray mass spectrometry measurement s (LC/ESI-MS). The subunits were separated by reversed-phase high-perf ormance liquid chromatography using a water/formic acid gradient. Of t he detergents used to solubilize the sample, the uncharged dodecylmalt oside and the positively charged dodecyltrimethylammoniumchloride had the most favorable influence on the separation and mass measurement by ESI-MS. For the first time the rather hydrophilic IIAB(Man) (34 920.8 +/- 6.7 Da) and the transmembrane IIDMan (31 909.4 +/- 18.2 Da) subun its could be determined with a mass accuracy in the range of 0.01 to 0 .05%, On the other hand no appropriate conditions could be found to de termine the precise mass of IICMan experimentally. (C) 1997 by John Wi ley & Sons, Ltd.