Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

Zhou, Mowei; Lantz, Carter; Brown, Kyle A.; Ge, Ying; Paša‐Tolić, Ljiljana; Loo, Joseph A.; Lermyte, Frederik

doi:10.1039/d0sc04392c

Cited by 109 publications

(138 citation statements)

References 176 publications

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“…This ability to unambiguously elucidate the structure of a specific S-RBD glycoform with high accuracy and quantify its relative abundance demonstrates the distinct advantages of this native top-down MS strategy over glycopeptide-based bottom-up MS approaches. 47,62 Together, this proteoform-resolved intact glycoprotein analysis strategy enables the simultaneous characterization of O-glycan structures of the S-RBD and its microheterogeneity, including structure and relative molecular abundance (Figure 6C). Importantly, the accurate determination of the relative abundance of intact glycoforms provides the technical foundation to understand the functional significance of these distinct glycoforms of S protein in the future.…”

Section: This Native Top-down Ms Approach Enables the Proteoform-resolved Characterization Of S-rbd O-glycoforms To Identify And Charactementioning

confidence: 99%

“…[40][41][42] In contrast, by combining glycoproteomics with the top-down MS approach, which preserves the intact glycoprotein enabling high-resolution proteoform-resolved analysis, [43][44][45] we could achieve the simultaneous characterization of the molecular structures, the site specificity, and the relative abundance of various glycoforms. Furthermore, by integrating native MS, which has recently emerged as a powerful structural biology tool to study protein structure-function relationships, [46][47][48][49][50][51][52] with trapped ion mobility spectrometry (TIMS), [53][54][55] we can investigate the gas phase structural variants to achieve the direct quantification of individual glycoproteoforms.…”

Section: Introductionmentioning

confidence: 99%

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Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

Roberts

Mann

Melby

et al. 2021

Preprint

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation is strongly implicated in altering viral binding/function and infectivity. However, the structures and relative abundance of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis. Here, we report the complete structural characterization of intact O-glycan proteoforms using native top-down mass spectrometry (MS). By combining trapped ion mobility spectrometry (TIMS), which can separate the protein conformers of S-RBD and analyze their gas phase structural variants, with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analysis, the O-glycoforms of the S-RBD are comprehensively characterized, so that seven O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that native top-down MS can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general.

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Section: This Native Top-down Ms Approach Enables the Proteoform-resolved Characterization Of S-rbd O-glycoforms To Identify And Charactementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

Roberts

Mann

Melby

et al. 2021

Preprint

Self Cite

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“…Classic native MS can be utilized to get a general understanding about complex stoichiometry and PTMs. Nevertheless, standard native MS falls short of really understanding the connectivity of subunits and binding location [ 12 ]. Therefore, advanced native MS workflows for in-depth analysis of the primary to quaternary structure were used.…”

Section: Top-down Methodsmentioning

confidence: 99%

“…In bottom-up-based methods, proteins are proteolytically digested into peptides prior to their injection into the mass spectrometer, allowing an analysis of complex specimens from different biological material. Conversely, in top-down methods intact proteins are analyzed, making these workflows particularly suited for the analysis of different, co-existing post-translational modifications (PTMs) of a protein or protein complex [ 4 , 12 ]. Here we review current approaches, in particular proteomics-based datasets generated in the past 10 years that have been used to identify, profile, or characterize novel elusive aspects of RNA virus biology, including replication mechanisms and interaction with their hosts.…”

Section: Introductionmentioning

confidence: 99%

Top-Down and Bottom-Up Proteomics Methods to Study RNA Virus Biology

Simanjuntak

Schamoni-Kast

Grün

et al. 2021

Viruses

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RNA viruses cause a wide range of human diseases that are associated with high mortality and morbidity. In the past decades, the rise of genetic-based screening methods and high-throughput sequencing approaches allowed the uncovering of unique and elusive aspects of RNA virus replication and pathogenesis at an unprecedented scale. However, viruses often hijack critical host functions or trigger pathological dysfunctions, perturbing cellular proteostasis, macromolecular complex organization or stoichiometry, and post-translational modifications. Such effects require the monitoring of proteins and proteoforms both on a global scale and at the structural level. Mass spectrometry (MS) has recently emerged as an important component of the RNA virus biology toolbox, with its potential to shed light on critical aspects of virus–host perturbations and streamline the identification of antiviral targets. Moreover, multiple novel MS tools are available to study the structure of large protein complexes, providing detailed information on the exact stoichiometry of cellular and viral protein complexes and critical mechanistic insights into their functions. Here, we review top-down and bottom-up mass spectrometry-based approaches in RNA virus biology with a special focus on the most recent developments in characterizing host responses, and their translational implications to identify novel tractable antiviral targets.

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“…To better capture the inhibitor binding profile, we also applied surface-induced dissociation (SID), which involves collisions with a surface. Distinct from CID, SID is considered a "fast heating" process, which generally induces less unfolding and allows for better ligand preservation on released subunits [25,26].…”

Section: Native Mass Spectrometry (Ms) and Molecular Docking Of Nsp15mentioning

confidence: 99%

High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity

Choi

Zhou

Shek

et al. 2021

Preprint

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SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 μM range in vitro. Furthermore, Exebryl-1, a β-amyloid anti-aggregation molecule for Alzheimer’s therapy, was shown to have antiviral activity between 10 to 66 μM, in VERO, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral.Author summaryDrugs to treat COVID-19 are urgently needed. To address this, we searched libraries of drugs and drug-like molecules for inhibitors of an essential enzyme of the virus that causes COVID-19, SARS-CoV-2 nonstructural protein (nsp)15. We found several molecules that inhibited the nsp15 enzyme function and one was shown to be active in inhibiting the SARS-CoV-2 virus. This demonstrates that searching for SARS-CoV-2 nsp15 inhibitors can lead inhibitors of SARS-CoV-2, and thus therapeutics for COVID-19. We are currently working to see if these inhibitors could be turned into a drug to treat COVID-19.

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Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

Abstract: Top-down mass spectrometry techniques break up native proteins and complexes to reveal all levels of structural information.

Cited by 109 publications

References 176 publications

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

Top-Down and Bottom-Up Proteomics Methods to Study RNA Virus Biology

High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity

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