High-fidelity mass analysis unveils heterogeneity in intact ribosomal particles

Waterbeemd, Michiel van de; Fort, Kyle L.; Boll, Dmitriy; Reinhardt-Szyba, Maria; Routh, Andrew; Makarov, Alexander; Heck, Albert J. R.

doi:10.1038/nmeth.4147

Cited by 158 publications

(220 citation statements)

References 43 publications

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“…It does require fluorophore labeling which can be limited in throughput, by epitope occlusion, or lead to artifacts if fluorescent proteins as used. Alternatively, top-down mass spectrometry approaches, where intact proteins or complexes can be analyzed to determine structural and conformational information have also begun to identify altered ribosome compositions [76]. This ability to precisely define specific, individual ribosome conformations, will prove invaluable for proving true RP heterogeneity within single cells.…”

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confidence: 99%

Ribosome stoichiometry: from form to function.

Emmott

Jovanović

Slavov

2018

Preprint

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The existence of eukaryotic ribosomes with distinct ribosomal protein (RP) stoichiometry and regulatory roles in protein synthesis been speculated for over sixty years. Recent advances in mass spectrometry and high throughput analysis have begun to identify and characterize distinct ribosome stoichiometry in yeast or mammalian systems. In addition to RP stoichiometry, ribosomes play host to a vast array of protein modifications, effectively expanding the number of human RPs from 80 to many thousands of distinct proteoforms. Is it possible these proteoforms combine to function as a 'ribosome code' to tune protein synthesis? We outline the specific benefits that translational regulation by specialized ribosomes can offer and discuss the means and methodologies available to correlate and characterize RP stoichiometry with function. We highlight previous research with a focus on formulating hypotheses that can guide future experiments and crack the 'ribosome code'.Ribosomes, the cellular machinery of protein synthesis, are present at up to ten million copies per cell in mammals. Despite their abundance and the wide array of known modifications to both ribosomal proteins (RPs) and rRNA, study of the direct role of the ribosome in tuning cellular translation has until recently taken a back seat to posttranscriptional regulation at the level of translation initiation. The hypothesis that ribosomes actively regulate protein synthesis as part of normal development and physiology dates back to the 1950s [1]. In the ensuing decades, numerous albeit inconclusive, observations have supported this hypothesis, and a subset of those are shown in Figure 1, color-coded by the type of evidence.For many years, the dominant 'abundance' model of translational regulation by the ribosome suggested a limited role for ribosomes in translation regulation [2]. In this model, if ribosomes have different initiation affinity for different transcripts, a global decrease in the availability of free ribosomes selectively decreases the initiation rates of different transcripts to varying degrees [2]. This mechanism, recently reviewed by Mills and Green [4], relies on the non-linear dependence of translation initiation on free ribosomes and applies quite generally. Indeed, recent research from the Sankaran lab suggested this mechanisms could explain the failure of erythroid lineage commitment seen with Diamond-Blackfan anaemia [5] 2 by the abundance model is limited in magnitude by the changes of total ribosomal content and in flexibility since it provides unidirectional regulation for all proteins.The concept of ribosome specialization In the 'specialized' ribosome model, ribosomes do not possess constant structure or composition (Figure 2A, B), and instead exhibit altered stoichiometry of what were previously thought to represent 'core' ribosomal proteins (Figure 1) [7,8]. In this model, different ribosomal compositions are functional and have specific roles in translation. Specialized ribosomes could co-exist within cells, or between ...

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confidence: 99%

Ribosome stoichiometry: from form to function.

Emmott

Jovanović

Slavov

2018

Preprint

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“…While DESI has also been adapted to study large biomolecules, via the mixing of ES droplets and solution in a process known as liquid DESI,5, 6 it has not yet been applied to proteins deposited on surfaces and desorbed in solutions that retain their native state interactions. Despite considerable progress in applications of non‐denaturing or native MS (nMS) of soluble7, 8, 9 and membrane embedded proteins10 the possibility of effectively “lifting” intact complexes from surfaces is desirable since many high throughput technologies then become accessible 11. Moreover the lipid distribution in natural membranes is essentially planar and asymmetric with varying spatial and temporal arrangements in the vicinity of embedded protein complexes 12.…”

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confidence: 99%

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

et al. 2017

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Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non‐covalent interactions we exploit the surface to capture a rapid turnover enzyme–substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high‐throughput ligand screening of some of the most challenging drug targets including GPCRs.

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“…Such complexes may be combinations of individual proteoforms of the same protein, or they may be combinations of proteins stemming from different genes. Alternatively, interactions between different proteins and RNA, such as ribosomal RNA (rRNA), result in the proper assembly of entire organelles, such as ribosomes [7]. In each case, each macromolecular complex represents an additional level of complexity and structural heterogeneity.…”

Section: Heterogeneity Of Proteinsmentioning

confidence: 99%

“…The ability to fine-tune collisional gas pressure in the HCD cell allows for the optimization of trapping and collisional cooling of large intact proteins, thereby improving S/N dramatically for large analytes. Several recent studies have involved analysis of intact mAbs, macromolecular assemblies of non-covalent protein complexes approaching molecular weights of 1.0 MDa, and even intact ribosomal subunits under native conditions using the Exactive EMR MS [7,134,201]. Lastly, the Exactive Plus EMR allows for all-ion fragmentation (AIF) in the HCD cell.…”

Section: Exactive Emr Msmentioning

confidence: 99%

“…For example, a modified Exactive Plus Orbitrap instrument was applied to distinguish between ATP-and ADP-bound states of the 0.8 MDa E. coli chaperonin GroEL, a fourteen subunit complex [134]. It was further demonstrated that a modified Exactive Plus Orbitrap instrument could be used for native MS experiments of substrates with molecular weights approaching 10.0 MDa while still possessing sufficient resolution and sensitivity to detect small changes to the large molecular mass of the multisubunit protein complex or macromolecular assembly [134,318]. Moreover, recent studies have shown that an Orbitrap-based instrument can efficiently perform ion isolation of the complex and fragmentation into individual monomeric subunits (MS 2 ) followed by subsequent isolation of subunit ions and fragmentation into backbone fragments (MS 3 ) [135,317].…”

Section: Introductionmentioning

confidence: 99%

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Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

Belov¹

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High-fidelity mass analysis unveils heterogeneity in intact ribosomal particles

Cited by 158 publications

References 43 publications

Ribosome stoichiometry: from form to function.

Ribosome stoichiometry: from form to function.

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

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