Structural mass spectrometry approaches to study the 20S proteasome

Ben‐Nissan, Gili; Vimer, Shay; Tarnavsky, Mark; Sharon, Michal

doi:10.1016/bs.mie.2018.12.029

“…We identified half-proteasome species (77), an 7 ring, and a 777 particle, as expected from the stacked four-ring structure of this complex. We also identified a subcomplex of the proteasome that was missing two  subunits (7775) 60,61 . Moreover, we identified a population of heterodimers corresponding in mass to PSMA2-PSMA6 (Fig.…”

Section: Sid Coupled To Im-ms Measurements Reflects 20s Proteasome Topologymentioning

confidence: 99%

“…Although native MS does not deliver structures at atomic resolution, its advantage lies in its rapid analysis, its low sample amount requirement and its ability to a provide insight into protein conformational dynamics and coexisting transient species in solution [29][30][31][32][33] . We applied a set of MS based approaches to dissect the distinct structural attributes of these highly related 20S proteasome complexes.…”

Section: Introductionmentioning

confidence: 99%

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

Vimer¹,

Nissan²,

Morgenstern³

et al. 2020

Preprint

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Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multi-level experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the T. acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (S. cerevisiae) and mammals (rat - R. norvegicus, rabbit - O. cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native Ms structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution three-dimensional structures, highly resembled that of the human complex. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size, and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the <a></a><a>PSMA7 </a>subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.

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“…The different 20S proteasomes were buffer-exchanged once into 150 mM ammonium acetate [39] and diluted to 3 μM. For binding assays, the buffer-exchanged MCP21 medium was diluted 10-fold with 150 mM ammonium acetate and mixed 1:1 with 3 μM of the different proteasome samples.…”

Section: Sample Preparationmentioning

confidence: 99%

Direct‐MS analysis of antibody‐antigen complexes

Vimer

¹

,

Ben‐Nissan

²

,

Marty

³

et al. 2021

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In recent decades, antibodies (Abs) have attracted the attention of academia and the biopharmaceutical industry due to their therapeutic properties and versatility in binding a vast spectrum of antigens. Different engineering strategies have been developed for optimizing Ab specificity, efficacy, affinity, stability and production, enabling systematic screening and analysis procedures for selecting lead candidates. This quality assessment is critical but usually demands time-consuming and labor-intensive purification procedures. Here, we harnessed the direct-mass spectrometry (direct-MS) approach, in which the analysis is carried out directly from the crude growth media, for the rapid, structural characterization of designed Abs. We demonstrate that properties such as stability, specificity and interactions with antigens can be defined, without the need for prior purification.

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“…20S proteasomes from rat livers, human HEK293 cells, and yeast cells were purified as described 61 .…”

Section: Sample Preparationmentioning

confidence: 99%

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

Vimer¹,

Nissan²,

Morgenstern³

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multi-level experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the T. acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (S. cerevisiae) and mammals (rat - R. norvegicus, rabbit - O. cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native Ms structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution three-dimensional structures, highly resembled that of the human complex. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size, and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the <a></a><a>PSMA7 </a>subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.

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Structural mass spectrometry approaches to study the 20S proteasome

Cited by 20 publications

References 90 publications

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

Direct‐MS analysis of antibody‐antigen complexes

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

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