In-Cell Labeling and Mass Spectrometry for Systems-Level Structural Biology

Chavez, Juan D.; Wippel, Helisa Helena; Tang, Xiaoting; Keller, Andrew; Bruce, James E.

doi:10.1021/acs.chemrev.1c00223

Cited by 21 publications

(26 citation statements)

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“…Structural information can also be obtained from solvent-accessibility foot-printing using chemical reactive species and later characterized by MS analysis. , Among the approaches using this rationale, HDX is the most popular, but it has not been extensively used with live cells. ,, Different methods were developed and applied in cells, ,, among which fast photochemical oxidation of proteins (FPOP) appears to be promising. ,, This technique generates chemical reactions on the microsecond time scale, which can reveal the solvent-accessible residues of thousands of proteins from about 10 million cells, − or from 10,000 Caenorhabditis elegans individuals. , While FPOP in vitro has been shown to be capable of characterizing epitope mapping, conformational changes, and even of yielding good protein structure predictions, , it is still in a development phase in cells. , …”

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

“…Structural information and drug binding characterization might be extracted from MS protein-protein interactomes obtained via biotinylation proximity assays, ,, even though these methods are biased toward IDPs . MS-derived structural information has also been obtained recently using cross-linking agents (disuccinimidyl-suberate or -sulfoxide) in cells ,,− or in purified mitochondria. , The identified cross-linked peptides inform about their intra- and intermolecular proximities in cells. These great achievements have been carried out upon 0.5–3 h long incubation times in PBS at room temperature.…”

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

“…Structural information can also be obtained from solventaccessibility foot-printing using chemical reactive species and later characterized by MS analysis. 837,838 Among the approaches using this rationale, HDX is the most popular, but it has not been extensively used with live cells. 539,838,839 Different methods were developed and applied in cells, 838,840,841 among which fast photochemical oxidation of proteins (FPOP) appears to be promising.…”

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

“…837,838 Among the approaches using this rationale, HDX is the most popular, but it has not been extensively used with live cells. 539,838,839 Different methods were developed and applied in cells, 838,840,841 among which fast photochemical oxidation of proteins (FPOP) appears to be promising. 838,842,843 This technique generates chemical reactions on the microsecond time scale, which can reveal the solvent-accessible residues of thousands of proteins from about 10 million cells, 844−847 or from 10,000 Caenorhabditis elegans individuals.…”

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

“…539,838,839 Different methods were developed and applied in cells, 838,840,841 among which fast photochemical oxidation of proteins (FPOP) appears to be promising. 838,842,843 This technique generates chemical reactions on the microsecond time scale, which can reveal the solvent-accessible residues of thousands of proteins from about 10 million cells, 844−847 or from 10,000 Caenorhabditis elegans individuals. 848,849 While FPOP in vitro has been shown to be capable of characterizing epitope mapping, conformational changes, and even of yielding good protein structure predictions, 842,850 it is still in a development phase in cells.…”

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

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In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

Theillet

2022

Chem. Rev.

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In-cell structural biology aims at extracting structural information about proteins or nucleic acids in their native, cellular environment. This emerging field holds great promises and is already providing new facts and outlooks of interest at both fundamental and applied levels. NMR spectroscopy has important contributions on this stage: it brings information on a broad variety of nuclei at the atomic scale, which ensures its great versatility and uniqueness. Here, we detail the methods, the fundamental knowledge and the applications in biomedical engineering related to in-cell NMR. We finally propose a brief overview of the main other techniques in the field (EPR, smFRET, cryo-ET…) to draw some advisable developments for in-cell NMR. In the era of large-scale screenings and deep learning, both accurate and qualitative experimental evidences are as essential as ever to understand the interior life of cells. In-cell structural biology by NMR spectroscopy can generate such a knowledge, and it does so at the atomic scale. This review is meant to deliver comprehensive but accessible information, with advanced technical details and reflections on the methods, the nature of the results and the future of the field. CONTENTS 5.1.1. In-cell EPR 5.1.2. In-cell FRET microscopy 5.1.3. Mass-spectrometry 5.1.4. Cryo-ET 5.2. The specific benefits of in-cell NMR 5.3. The future technical challenges of in-cell NMR 6. Conclusion Author Information

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Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

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In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

Theillet

2022

Chem. Rev.

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Using mass spectrometry‐based methods to understand amyloid formation and inhibition of alpha‐synuclein and amyloid beta

Wagner

Gross

2022

Mass Spectrometry Reviews

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Amyloid fibrils, insoluble β‐sheets structures that arise from protein misfolding, are associated with several neurodegenerative disorders. Many small molecules have been investigated to prevent amyloid fibrils from forming; however, there are currently no therapeutics to combat these diseases. Mass spectrometry (MS) is proving to be effective for studying the high order structure (HOS) of aggregating proteins and for determining structural changes accompanying protein–inhibitor interactions. When combined with native MS (nMS), gas‐phase ion mobility, protein footprinting, and chemical cross‐linking, MS can afford regional and sometimes amino acid spatial resolution of the aggregating protein. The spatial resolution is greater than typical low‐resolution spectroscopic, calorimetric, and the traditional ThT fluorescence methods used in amyloid research today. High‐resolution approaches can struggle when investigating protein aggregation, as the proteins exist as complex oligomeric mixtures of many sizes and several conformations or polymorphs. Thus, MS is positioned to complement both high‐ and low‐resolution approaches to studying amyloid fibril formation and protein–inhibitor interactions. This review covers basics in MS paired with ion mobility, continuous hydrogen‐deuterium exchange (continuous HDX), pulsed hydrogen‐deuterium exchange (pulsed HDX), fast photochemical oxidation of proteins (FPOP) and other irreversible labeling methods, and chemical cross‐linking. We then review the applications of these approaches to studying amyloid‐prone proteins with a focus on amyloid beta and alpha‐synuclein. Another focus is the determination of protein–inhibitor interactions. The expectation is that MS will bring new insights to amyloid formation and thereby play an important role to prevent their formation.

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Dissecting the structural heterogeneity of proteins by native mass spectrometry

2023

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A single gene yields many forms of proteins via combinations of posttranscriptional/posttranslational modifications. Proteins also fold into higher‐order structures and interact with other molecules. The combined molecular diversity leads to the heterogeneity of proteins that manifests as distinct phenotypes. Structural biology has generated vast amounts of data, effectively enabling accurate structural prediction by computational methods. However, structures are often obtained heterologously under homogeneous states in vitro. The lack of native heterogeneity under cellular context creates challenges in precisely connecting the structural data to phenotypes. Mass spectrometry (MS) based proteomics methods can profile proteome composition of complex biological samples. Most MS methods follow the “bottom‐up” approach, which denatures and digests proteins into short peptide fragments for ease of detection. Coupled with chemical biology approaches, higher‐order structures can be probed via incorporation of covalent labels on native proteins that are maintained at the peptide level. Alternatively, native MS follows the “top‐down” approach and directly analyzes intact proteins under nondenaturing conditions. Various tandem MS activation methods can dissect the intact proteins for in‐depth structural elucidation. Herein, we review recent native MS applications for characterizing heterogeneous samples, including proteins binding to mixtures of ligands, homo/hetero‐complexes with varying stoichiometry, intrinsically disordered proteins with dynamic conformations, glycoprotein complexes with mixed modification states, and active membrane protein complexes in near‐native membrane environments. We summarize the benefits, challenges, and ongoing developments in native MS, with the hope to demonstrate an emerging technology that complements other tools by filling the knowledge gaps in understanding the molecular heterogeneity of proteins.

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In-Cell Labeling and Mass Spectrometry for Systems-Level Structural Biology

Cited by 21 publications

References 265 publications

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

Using mass spectrometry‐based methods to understand amyloid formation and inhibition of alpha‐synuclein and amyloid beta

Dissecting the structural heterogeneity of proteins by native mass spectrometry

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