Mass Measurements of Increased Accuracy Resolve Heterogeneous Populations of Intact Ribosomes

Abstract

It is established that noncovalent complexes can be maintained both during and after electrospray and that assemblies of increasing size and complexity often lead to broadened peaks in mass spectra. This broadening arises from the tendency of large protein assemblies to form adducts with salts and is compounded when complexes are isolated directly from cells, without the full protein complement. To investigate the origins of this broadening in mass spectral peaks and to develop the optimal method for analyzing mass spectra of large protein complexes, we have carried out a systematic investigation of a series of noncovalent complexes representing a range of different sizes and architectures. We establish a positive correlation between peak width and the increased mass observed and show that this correlation is independent of the instrumental parameters employed. Using this relationship we show that we can determine masses of both 30S subunits and intact 2.3 MDa 70S ribosomes from Thermus thermophilus. The masses of both particles are consistent with multiple populations of ribosomes. To identify these various populations we combine simulated mass spectra of ribosomes, with and without the full protein complement, and estimate the extent of adducts from our study of known complexes. The results allow us to determine the contribution of the different subpopulations to the overall mass spectrum. We confirm the existence of these subpopulations using tandem mass spectrometry of intact 30S subunits. Overall, the results show that, rather than uniform particles, gas-phase ribosomes consist of a number of discrete populations. More generally, the results establish a rigorous procedure for accurate mass measurement and spectral analysis of heterogeneous macromolecular assemblies.

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

“…Charge states of both the 30S and 50S particles are partially resolved. The mass of the 30S subunit is consistent with loss of S1 and S6 as observed previously (35 Schematic representation of the L7/L12 exchange mechanism which takes place between ribosomes in the absence of factors (left) and in the ribosome-EF-G complex (right). The schematic shows all events that were considered in our model.…”

Gas-phase H/D exchange and collision cross sections of hemoglobin monomers, dimers, and tetramers

“…This is commonly seen with ESI-MS of protein-protein complexes and has been attributed to clustering with solvent molecules or other species [52][53][54][55]. In an attempt to reduce clustering of the tetramer peaks, 100 M hemoglobin was run in 25% methanol, as described by Simmons et al [32].…”

Ion mobility–mass spectrometry analysis of large protein complexes

“…These conditions often include increased pressures in the source region of the instrument and carefully reducing the amount of 'collisional heating' (also termed, 'activation') experienced by protein complex ions to avoid dissociation 38 . However, as discussed previously, some activation is usually employed to desolvate the protein complex ions and achieve optimum mass accuracy 39,40 . Figure 2d shows a series of mass spectra for TaHSP16.9 acquired using increasing accelerating voltage into the storage ion guide located before the IM separator.…”