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Analysis of Hydrophobic Proteins and Peptides by Electrospray Ionization Mass Spectrometry
Author(s) -
Schaller Johann,
Pellascio Bernhard Christian,
Schlunegger Urs Peter
Publication year - 1997
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(19970228)11:4<418::aid-rcm795>3.0.co;2-r
Subject(s) - chemistry , chromatography , formic acid , mass spectrometry , electrospray ionization , electrospray , protein mass spectrometry , tandem mass spectrometry
Bacterioopsin (BO) from Halobacterium halobium , as well as its V8‐protease and CNBr fragments from the C‐terminal region, were used to establish appropriate conditions for the mass determination of membrane proteins 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/) containing 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 , IIC Man and IID Man of the mannose transporter complex in E. coli were used to establish conditions for liquid chromatography/electrospray mass spectrometry measurements (LC/ESI‐MS). The subunits were separated by reversed‐phase high‐performance liquid chromatography using a water/formic acid gradient. Of the detergents used to solubilize the sample, the uncharged dodecylmaltoside 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 IID Man (31 909.4 ± 18.2 Da) subunits 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 determine the precise mass of IIC Man experimentally. © 1997 John Wiley & Sons, Ltd.