Mass spectrometry of ribosomes and ribosomal subunits

^*Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QT, United Kingdom; and ^†Howard Hughes Medical Institute, Cellular Biochemistry and Biophysics Program, Memorial-Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021

Communicated by Harry B. Gray, California Institute of Technology, Pasadena, CA (received for review January 21, 1998)

Abstract

Nanoflow electrospray ionization has been used to introduce intact Escherichia coli ribosomes into the ion source of a mass spectrometer. Mass spectra of remarkable quality result from a partial, but selective, dissociation of the particles within the mass spectrometer. Peaks in the spectra have been assigned to individual ribosomal proteins and to noncovalent complexes of up to five component proteins. The pattern of dissociation correlates strongly with predicted features of ribosomal protein–protein and protein–RNA interactions. The spectra allow the dynamics and state of folding of specific proteins to be investigated in the context of the intact ribosome. This study demonstrates a potentially general strategy to probe interactions within complex biological assemblies.

Footnotes

↵ ‡ Present address: Bristol-Meyers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492-7660.
↵ § Present address: Max Planck Institute für Biochemie, Am Klopferspitz 18A, D-82152 Martinsried, Germany.
↵ ¶ To whom reprint requests should be addressed. e-mail: chris.dobson{at}chem.ox.ac.uk.
↵ ‖ Although ribosomal subunit association generally is thought to require Mg²⁺, adducts with this cation reduce resolution and sensitivity of ESI mass spectra; we therefore excluded it from the buffers in the present study. Fluorescence measurements of ethidium bromide binding to intact ribosomes and isolated subunits have, however, led to the proposal that subunits interact even in the absence of Mg²⁺ (19).
↵ ** Globular proteins generally have higher mass/charge ratios than unfolded proteins (17). Similarly, assemblies of proteins have higher mass/charge ratios than their individual components; for the L7/L10/L12 complex, for example, the charge-state distribution is centered around z = 18, rather than z = 38, which would be predicted from the sum of the species from the component proteins. The lack of detectable ions for intact large complexes therefore is likely to arise in part from mass/charge ratios too high for detection in conventional spectrometers.
ABBREVIATION:

ESI,

electrospray ionization