Separating and visualising protein assemblies by means of preparative mass spectrometry and microscopy.
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.
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Justin L P
JL
Benesch
Brandon T
BT
Ruotolo
Douglas A
DA
Simmons
Nelson P
NP
Barrera
Nina
N
Morgner
Luchun
L
Wang
Helen R
HR
Saibil
Carol V
CV
Robinson
D018834:Chaperonin 60 / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D004926:Escherichia coli / Q000737:chemistry; D018625:Microscopy, Atomic Force / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D018834:Chaperonin 60; D046912:Multiprotein Complexes