Measurement of the molecular masses of hydrophilic and hydrophobic subunits of ATP synthase and complex I in a single experiment.

TitleMeasurement of the molecular masses of hydrophilic and hydrophobic subunits of ATP synthase and complex I in a single experiment.
Publication TypeJournal Article
Year of Publication2009
AuthorsCarroll, J, Fearnley, IM, Wang, Q, Walker, JE
JournalAnal Biochem
Volume395
Issue2
Pagination249-55
Date Published2009 Dec 15
ISSN1096-0309
KeywordsAnimals, ATP Synthetase Complexes, Cattle, Chromatography, Liquid, Electron Transport Complex I, Mass Spectrometry, Mitochondria, Heart, Molecular Weight, Protein Structure, Tertiary, Spectrometry, Mass, Electrospray Ionization
Abstract

The adenosine triphosphate (ATP) synthase and complex I in mitochondria are membrane-bound multisubunit assemblies of both hydrophilic and hydrophobic proteins. Hitherto, the mass spectrometric measurement of their molecular masses has required that many of the hydrophobic proteins be analyzed separately from the other components in two different experiments. Here we describe a procedure that allows the molecular masses of all, or nearly all, of the subunits of each complex to be measured in a single experiment. The key feature is a mobile phase, in which hydrophilic and hydrophobic components remain soluble, that is compatible with reverse phase chromatography. In this way, the masses of all 17 subunits of bovine ATP synthase, 14 of the 17 subunits of the enzyme from Saccharomyces cerevisiae, 42 of the 45 subunits of bovine complex I, and all 28 of the subunits of bovine subcomplex Ialpha were measured. The method was used to characterize the subunits of ATP synthases and complexes I from a variety of species and to follow the progress of mild trypsinolysis of ATP synthase. It could be applied to other respiratory and photosynthetic complexes and, in general, to any protein complex that contains both hydrophilic and hydrophobic subunits.

DOI10.1016/j.ab.2009.08.006
Alternate JournalAnal. Biochem.
Citation Key10.1016/j.ab.2009.08.006
PubMed ID19679095
Grant ListMC_U105663148 / / Medical Research Council / United Kingdom