Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry.
The growing number of applications to determine the stoichiometry, interactions and even subunit architecture of protein complexes from mass spectra suggests that some general guidelines can now be proposed. In this protocol, we describe the necessary steps required to maintain interactions between subunits in the gas phase. We begin with the preparation of suitable solutions for electrospray (ES) and then consider the transmission of complexes through the various stages of the mass spectrometer until their detection. Subsequent steps are also described, including the dissociation of these complexes into multiple subcomplexes for generation of interaction networks. Throughout we highlight the critical experimental factors that determine success. Overall, we develop a generic protocol that can be carried out using commercially available ES mass spectrometers without extensive modification.
Hernández
Helena
H
Department of Chemistry, Cambridge University, Lensfield Road, Cambridge CB2 1EW, UK.
Robinson
Carol V
CV
D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D046912:Multiprotein Complexes