Advances in mass spectrometry‐based footprinting of membrane proteins

Li, Weikai; Gross, Michael L.

doi:10.1002/pmic.202100222

Cited by 5 publications

(7 citation statements)

References 105 publications

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“…Similar to FPOP, X‐ray footprinting (XFMS) utilizes hydroxyl radical labeling to determine protein HOS (Bavro et al, 2015; Maleknia et al, 2001; J. Sun et al, 2022; Xu & Chance, 2007; Gupta; Guan & Chance, 2005; Takamoto & Chance, 2006). At the National Synchrotron Light Source and the Advanced Light Source, photons of 3–30 keV are produced and can interact with water to create hydroxyl radicals (see Scheme 3) (Bavro et al, 2015; Guan & Chance, 2005; Gupta et al, 2016; Maleknia et al, 2001; J.…”

Section: Methods For Studying Aggregating Proteinsmentioning

confidence: 99%

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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Section: Methods For Studying Aggregating Proteinsmentioning

confidence: 99%

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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“…Recent advances have been made that greatly expand the utility of covalent labeling reagents for mapping interacting surfaces and for reporting on conformational changes in soluble proteins. ,− For example, new reagents have been developed that can react with aromatic sidechains within membrane proteins and on hydrophobic patches, − and a handful of recently developed methods are aimed at measuring proteome-wide conformational changes. − In this report, while we set out to exploit the azido radical as a simpler means of general covalent protein labeling and subsequent derivatization, the product of these experiments is also a method for covalently modifying protein with the azido radical that captures and identifies locations of noncovalent azide binding. With our method, azide may present a unique, enrichable method to add to the existing toolbox.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, it appears azide behaves similarly to the covalent labeling reagent diethylpyrocarbonate (DEPC), which has been reported to modify residues found in transmembrane domains and in soluble proteins, within structured hydrophobic patches. The reactivity of DEPC with soluble proteins is destroyed when the higher-order protein structure is greatly reduced via proteolytic digestion, ,− and suggests DEPC can empirically identify amino acids present in structured hydrophobic protein microdomains. Underscoring the need for empirical methods to identify hydrophobic microdomains, recent computational studies have indicated that hydrophilic amino acid sidechains are as likely to be present in these hydrophobic microdomains as are the hydrophobic sidechains, confounding the prediction of such patches from the primary sequence .…”

Section: Introductionmentioning

confidence: 99%

Radical-Mediated Covalent Azidylation of Hydrophobic Microdomains in Water-Soluble Proteins

et al. 2023

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Hydrophobic microdomains, also known as hydrophobic patches, are essential for many important biological functions of water-soluble proteins. These include ligand or substrate binding, protein–protein interactions, proper folding after translation, and aggregation during denaturation. Unlike transmembrane domains, which are easily recognized from stretches of contiguous hydrophobic sidechains in amino acids via primary protein sequence, these three-dimensional hydrophobic patches cannot be easily predicted. The lack of experimental strategies for directly determining their locations hinders further understanding of their structure and function. Here, we posit that the small triatomic anion N3 – (azide) is attracted to these patches and, in the presence of an oxidant, forms a radical that covalently modifies C–H bonds of nearby amino acids. Using two model proteins (BSA and lysozyme) and a cell-free lysate from the model higher plant Arabidopsis thaliana, we find that radical-mediated covalent azidylation occurs within buried catalytic active sites and ligand binding sites and exhibits similar behavior to established hydrophobic probes. The results herein suggest a model in which the azido radical is acting as an “affinity reagent” for nonaqueous three-dimensional protein microenvironments and is consistent with both the nonlocalized electron density of the azide moiety and the known high reactivity of azido radicals widely used in organic chemistry syntheses. We propose that the azido radical is a facile means of identifying hydrophobic microenvironments in soluble proteins and, in addition, provides a simple new method for attaching chemical handles to proteins without the need for genetic manipulation or specialized reagents.

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“…MS-based footprinting is a valuable tool for studying the interactions of MPs with other molecules and their conformational changes. Some reviews discuss covalent labeling coupled with MS [ 39 ] and the fast footprinting of MPs [ 40 ]. In this review, we focus on several advanced footprinting methods coupled with MS for (i) membrane-associated proteins, (ii) extramembrane domains, and (iii) transmembrane domains, showing how methods can be developed to probe various domains of the protein.…”

Section: Chemical Footprintingmentioning

confidence: 99%

Advances in Mass Spectrometry on Membrane Proteins

Yang

Gross

2023

Membranes

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Understanding the higher-order structure of membrane proteins (MPs), which are vital for numerous biological processes, is crucial for comprehending their function. Although several biophysical approaches have been used to study the structure of MPs, limitations exist owing to the proteins’ dynamic nature and heterogeneity. Mass spectrometry (MS) is emerging as a powerful tool for investigating membrane protein structure and dynamics. Studying MPs using MS, however, must meet several challenges including the lack of stability and solubility of MPs, the complexity of the protein–membrane system, and the difficulty of digestion and detection. To meet these challenges, recent advances in MS have engendered opportunities in resolving the dynamics and structures of MP. This article reviews achievements over the past few years that enable the study of MPs by MS. We first introduce recent advances in hydrogen deuterium exchange and native mass spectrometry for MPs and then focus on those footprinting methods that report on protein structure.

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Advances in mass spectrometry‐based footprinting of membrane proteins

Cited by 5 publications

References 105 publications

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

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

Radical-Mediated Covalent Azidylation of Hydrophobic Microdomains in Water-Soluble Proteins

Advances in Mass Spectrometry on Membrane Proteins

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