Detection and selective dissociation of intact ribosomes in a mass spectrometer

Abstract

Intact Escherichia coli ribosomes have been projected into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques. Species with mass͞charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the stability of intact ribosomes was investigated and found to increase as a result of cross-linking ribosomal proteins to the rRNA. By lowering the Mg 2؉ concentration in solutions containing ribosomes the particles were found to dissociate into 30S and 50S subunits. The resolution of the charge states in the spectrum of the 30S subunit enabled its mass to be determined as 852,187 ؎ 3,918 Da, a value within 0.6% of that calculated from the individual proteins and the 16S RNA. Further dissociation into smaller macromolecular complexes and then individual proteins could be induced by subjecting the particles to increasingly energetic gas phase collisions. The ease with which proteins dissociated from the intact species was found to be related to their known interactions in the ribosome particle. The results show that emerging mass spectrometric techniques can be used to characterize a fully functional biological assembly as well as its isolated components. In recent years mass spectrometry has become the method of choice for a number of important aspects of experimental structural biology. These include: the characterization of the stability and folding behavior of proteins under a wide range of conditions (1-3), the identification of subpicomole quantities of proteins from two-dimensional gels (4), the de novo sequencing of peptides and subsequent cloning of novel proteins (5), and the analysis of the components of single vesicles (6). These applications have driven mass spectrometry to new levels of detection from a range of complex biological matrices. In addition, highresolution ion cyclotron resonance mass spectrometry has enabled the isolation of individual ions from polyethylene glycol (7) and DNA (8), with masses in excess of 10 8 Da. As well as being able to characterize highly charged polymers, it has been possible to detect signals from noncovalent complexes of small molecule ligands bound to proteins and of multiprotein complexes (9, 10). As the size of such assemblies increases, however, the number of charges acquired during the electrospray process increases less rapidly than the total mass. This phenomenon has been attributed to ionic interactions in the intermolecular interfaces and the appropriation of negatively charged counterions from the volatile buffers used in the analysis of such species (11). The net result of these effects is that large multimolecular complexes, such as the 2.3-MDa ribosome, have been outside the mass range of conventional mass spectrometers. In previous mass spectrometry experiments disruption of the ribosome enabled the identification of the contact sites between ribosomal proteins and RNA (12) and a novel protein component from the Saccharomyces cerevsiae ribosome (13). In add...

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

“…Some of the more successful approaches for characterizing ribosomes include crosslinking [1][2][3][4], cryo-electron microscopy (cryo-EM) [5][6][7], tritium bombardment [8], nuclear magnetic resonance (NMR) [9] and electrospray mass spectrometry [10][11][12][13]. One of the most successful approaches has been the development of X-ray crystallography within the past several years, wherein high-resolution crystal structures of ribosomal subunits or intact ribosomes have been obtained that provide detailed structural information at the atomic level [14][15][16][17][18].…”

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

“…To maintain the integrity of the 70S particles, a high [Mg 2+ ] is required together with a low ionic strength (28,34). From solutions containing 5 mM Mg 2+ , mass spectra confirm the presence of the intact 70S assembly (Figure 1, panel A) (28).…”

“…A recent X-ray structure of EF-G bound to the ribosome in the posttranslocational state demonstrates large scale movements in response to EF-G binding and reveals that a copy of an L12 CTD makes contact with EF-G (24), as suggested by previous cryo-electron microscopy data (27). The effects of this L12 CTD-EF-G interaction on the process of subunit exchange are currently unknown.We have shown previously that intact ribosomes and their subunits can be observed by MS (3,6,28,29), and that the intact stalk complex dissociates from the large subunit while retaining its non-covalent interactions (3,6,11). This allows us to study the composition of the stalk, without prior separation in solution, within the context of the intact ribosome.…”

“…We have shown previously that intact ribosomes and their subunits can be observed by MS (3,6,28,29), and that the intact stalk complex dissociates from the large subunit while retaining its non-covalent interactions (3,6,11). This allows us to study the composition of the stalk, without prior separation in solution, within the context of the intact ribosome.…”

A eole for the MS analysis of nucleic acids in the post-genomics age

“…In the absence of covalent fragmentation, the order by which bound subunits dissociate as discrete products may reveal their spatial situation within the complex of interest. Indeed, submitting intact ribosomes and their constitutive subunits to collisional activation induced the progressive release of protein units according to their affinity for each other and for the RNA components, but also according to their placement relative to the surface of the initial precursor ion [138,174]. In similar fashion, the dissociation of RNA-RNA pairing interactions was employed to interrogate the conformational state of assemblies folding into alternative isomeric forms [175].…”