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Many multi-protein assemblies exhibit characteristics which hamper their structural and dynamical characterization. These impediments include low copy number, heterogeneity, polydispersity, hydrophobicity, and intrinsic disorder. It is becoming increasingly apparent that both novel and hybrid structural biology approaches need to be developed to tackle the most challenging targets. Nanoelectrospray mass spectrometry has matured over the last decade to enable the elucidation of connectivity and composition of large protein assemblies. Moreover, comparing mass spectrometry data with transmission electron microscopy images has enabled the mapping of subunits within topological models. Here we describe a preparative form of mass spectrometry designed to isolate specific protein complexes from within a heterogeneous ensemble, and to 'soft-land' these target complexes for ex situ imaging. By building a retractable probe incorporating a versatile target holder, and modifying the ion optics of a commercial mass spectrometer, we show that we can steer the macromolecular ion beam onto a target for imaging by means of transmission electron microscopy and atomic force microscopy. Our data for the tetradecameric chaperonin GroEL show that not only are the molecular volumes of the landed particles consistent with the overall dimensions of the complex, but also that their gross topological features can be maintained.

Original publication

DOI

10.1016/j.jsb.2010.03.004

Type

Journal article

Journal

J Struct Biol

Publication Date

11/2010

Volume

172

Pages

161 - 168

Keywords

Chaperonin 60, Escherichia coli, Microscopy, Atomic Force, Microscopy, Electron, Transmission, Multiprotein Complexes, Spectrometry, Mass, Electrospray Ionization