In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in &lt;em&gt;Caenorhabditis elegans&lt;/em&gt;

Espino, Jessica A.; Jones, Lisa M.

doi:10.3791/60910

Cited by 7 publications

(8 citation statements)

References 9 publications

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“…One area in which HRF-MS is progressing more quickly is moving the workflow in cell, with both the membrane permeable hydrogen peroxide and laser based FPOP methods being applied successfully. This approach facilitates protein labeling within organelles and, in the case of Caenorhabditis elegans, within whole organisms, enabling structural interrogation under native conditions. − …”

Section: Structural Mass Spectrometry Methodsmentioning

confidence: 99%

“…This approach facilitates protein labelling within organelles and, in the case of C. elegans, within whole organisms, enabling structural interrogation under native conditions. [110][111][112]…”

Section: Hydroxyl Radical Footprinting-mass Spectrometry (Hrf-ms)mentioning

confidence: 99%

“…65,67,322 The FPOP derivative of HRF-MS has had similar in cell success over recent years, with a 2019 paper from Espino and Jones applying technique in vivo to determine protein solvent accessibility in C. elegans. [110][111][112] Given the prevalent use of C. elegans as a model organism, development of this workflow has exciting potential for the future study of disease mechanisms. The development of in vivo structural MS, however, is not limited to the peptide-based techniques, as developments have also been made in an attempt to bring protein-based methods into cells.…”

Section: Moving Structural Biology Towards In Cell Analysismentioning

confidence: 99%

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Integration of Mass Spectrometry Data for Structural Biology

2021

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Mass spectrometry (MS) is increasingly being used to probe the structure and dynamics of proteins and the complexes they form with other macromolecules. There are now several specialised MS methods each with unique sample preparation, data acquisition and data processing protocols. Collectively these methods are referred to as structural MS and include, crosslinking-, hydrogen deuterium exchange-, hydroxyl radical footprinting-native, ion mobility-and top-down MS. Each of these provides a unique type of structural information, ranging from composition and stoichiometry through to residue level proximity and solvent accessibility. Structural MS has proved particularly beneficial in studying protein classes for which analysis by classic structural biology techniques proves challenging, such as glycosylated or intrinsically disordered proteins. To capture the structural details for a particular system, especially larger multiprotein complexes, more than one structural MS method with other structural and biophysical techniques is often required. Key to integrating these diverse data are computational strategies and software solutions to facilitate this process.We provide a background to the structural MS methods and briefly summarise other structural methods and how these are combined with MS. We then describe current state of the art approaches for the integration of structural MS data for structural biology. We quantify how often these methods are used together and provide examples where such combinations have been fruitful. To illustrate the power of integrative approaches, we discuss progress in solving the structures of the proteasome and the nuclear pore complex. We also discuss how information from structural MS, particularly pertaining to protein dynamics is not currently utilised in integrative workflows and how such information can provide a more accurate picture of the systems studied. We conclude by discussing new developments in the MS and computational fields that will further enable in-cell structural studies.

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Section: Structural Mass Spectrometry Methodsmentioning

confidence: 99%

Section: Hydroxyl Radical Footprinting-mass Spectrometry (Hrf-ms)mentioning

confidence: 99%

Section: Moving Structural Biology Towards In Cell Analysismentioning

confidence: 99%

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Integration of Mass Spectrometry Data for Structural Biology

2021

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“…The procedure was performed with minor modifications as previously described . Prior to IV-FPOP, worms were kept separate from 200 mM hydrogen peroxide and the indicated CPE, mixed using a homemade flow system, and incubated for approximately 30 s just prior to IV-FPOP. Samples were flowed through a 250 μm i.d.…”

Section: Methodsmentioning

confidence: 99%

Chemical Penetration Enhancers Increase Hydrogen Peroxide Uptake in C. elegans for In Vivo Fast Photochemical Oxidation of Proteins

2020

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Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method that covalently labels solvent-accessible amino acids by photolysis of hydrogen peroxide. Recently, we expanded the use of FPOP for in vivo (IV-FPOP) covalent labeling in C. elegans. In initial IV-FPOP studies, 545 proteins were oxidatively modified in all body systems within the worm. Here, with the use of chemical penetration enhancers (CPEs), we increased the number of modified proteins as well as the number of modifications per protein to gain more structural information. CPEs aid in the delivery of hydrogen peroxide inside C. elegans by disturbing the highly ordered lipid bilayer of the worm cuticle without affecting worm viability. IV-FPOP experiments performed using the CPE azone showed an increase in oxidatively modified proteins and peptides. This increase correlated with greater hydrogen peroxide uptake by C. elegans quantified using a chemical fluorophore demonstrating the efficacy of using CPEs with IV-FPOP. Mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD019290.

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“…More recently, Espino and Jones developed the in vivo FPOP (IV-FPOP) method using the nematode Caenorhabditis elegans as biological model (Figure ). , The optimization of the IV-FPOP method, including changes in the H 2 O 2 concentration and in the microfluidic system, with LC-MS analysis, allowed for the identification of more than 500 oxidized proteins from different anatomical regions of the worm. Besides being a model system for the study of human diseases, C. elegans has the ability to intake H 2 O 2 and a transparent body that enables the laser light to penetrate the animal and, therefore making the IV-FPOP application feasible.…”

Section: Ms Structural Biology In Cellsmentioning

confidence: 99%

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

et al. 2021

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Biological systems have evolved to utilize proteins to accomplish nearly all functional roles needed to sustain life. A majority of biological functions occur within the crowded environment inside cells and subcellular compartments where proteins exist in a densely packed complex network of protein–protein interactions. The structural biology field has experienced a renaissance with recent advances in crystallography, NMR, and CryoEM that now produce stunning models of large and complex structures previously unimaginable. Nevertheless, measurements of such structural detail within cellular environments remain elusive. This review will highlight how advances in mass spectrometry, chemical labeling, and informatics capabilities are merging to provide structural insights on proteins, complexes, and networks that exist inside cells. Because of the molecular detection specificity provided by mass spectrometry and proteomics, these approaches provide systems-level information that not only benefits from conventional structural analysis, but also is highly complementary. Although far from comprehensive in their current form, these approaches are currently providing systems structural biology information that can uniquely reveal how conformations and interactions involving many proteins change inside cells with perturbations such as disease, drug treatment, or phenotypic differences. With continued advancements and more widespread adaptation, systems structural biology based on in-cell labeling and mass spectrometry will provide an even greater wealth of structural knowledge.

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In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in <em>Caenorhabditis elegans</em>

Cited by 7 publications

References 9 publications

Integration of Mass Spectrometry Data for Structural Biology

Integration of Mass Spectrometry Data for Structural Biology

Chemical Penetration Enhancers Increase Hydrogen Peroxide Uptake in C. elegans for In Vivo Fast Photochemical Oxidation of Proteins

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

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