Dissociation of Intact Escherichia coli Ribosomes in a Mass Spectrometer

Abstract

We used mass spectrometry to identify proteins that are released in the gas phase from Escherichia coli ribosomes in response to a range of different solution conditions and cofactor binding. From solution at neutral pH the spectra are dominated by just 4 of the 54 ribosomal proteins (L7/L12, L11, and L10). Lowering the pH of the solution leads to the gas phase dissociation of four additional proteins as well as the 5 S RNA. Replacement of Mg 2؉ by Li ؉ ions in solutions of ribosomes induced the dissociation of 17 ribosomal proteins. Correlation of these results with available structural information for ribosomes revealed that a relatively high interaction surface area of the protein with RNA was the major force in preventing dissociation. By using the proteins that dissociate to probe their interactions with RNA, we examined different complexes of the ribosome formed with the elongation factor G and inhibited by fusidic acid or thiostrepton. Mass spectra recorded for the fusidic acid-inhibited complex reveal subtle changes in peak intensity of the proteins that dissociate. By contrast gas phase dissociation from the thiostrepton-inhibited complex is markedly different and demonstrates the presence of L5 and L18, two proteins that interact exclusively with the 5 S RNA. These results allow us to propose that the ribosome elongation factor-G complex inhibited by thiostrepton, but not fusidic acid, involves destabilization of 5 S RNA-protein interactions.

Decharging of Globular Proteins and Protein Complexes in Electrospray

“…[1][2][3] In particular, electrospray ionization (ESI) has emerged as a powerful technique for generating ions of complex macromolecular species, in which the globular structure is at least partially retained by the ions. [4] By maintaining the proteins in a physiologically relevant buffer amenable to electrospray (e.g., aqueous ammonium acetate) prior to the ionization process, even noncovalent protein assemblies such as protein oligomers, [5][6][7][8] ribosomes, [9,10] and small viruses [11,12] can be transferred intact into the gas phase. The retention of globular shape by ions in the gas phase has been demonstrated by ion mobility measurements, in which protein ions with a limited number of charges exhibit collisional cross-sections comparable to those calculated from their crystal structures.…”

Surface-induced dissociation of small molecules, peptides, and non-covalent protein complexes

“…Through the contributions of a number of different research groups, numerous non-covalent assemblies have now been investigated in the gas phase [111][112][113][114][115][116][117]. The wide array of non-covalent systems that have been studied include complexes as simple as small protein-protein dimers and assemblies as daunting as the intact 70S ribosome, a heterogeneous 2 MDa complex composed of several RNA and over 50 different proteins [113,116]. Regardless of the complex, analysis by mass spectrometry has typically required nanospray ionization of the analyte from "near-physiological" (i.e., neutral pH) conditions.…”

Heptameric (L12)6/L10 rather than canonical pentameric complexes are found by tandem MS of intact ribosomes from thermophilic bacteria

“…In the 5.5-Å 70S structure of ribosomes from Thermus thermophilus density could not be assigned to L10, and only two of the four L7͞L12 proteins that have been proposed for Escherichia coli (5) were tentatively placed at the base of the stalk (6). Interestingly, the stalk complex is readily studied by MS where dissociation of proteins is governed primarily by the extent of protein-RNA interaction (7).…”

“…Although not readily applied to MDa particles such as ribosomes, the dissociation of individual proteins and stalk complexes from the intact particle has been shown (7,9,10). Such spectra are extremely difficult to interpret in part because of the number of proteins (Ͼ50), their numerous posttranslational modifications, and the presence of ancillary proteins and RNA.…”