Validation of the Applicability of In-Cell Fast Photochemical Oxidation of Proteins across Multiple Eukaryotic Cell Lines

Kaur, Upneet; Johnson, Danté T.; Jones, Lisa M.

doi:10.1021/jasms.0c00014

Cited by 15 publications

(19 citation statements)

References 46 publications

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“…532,817,818 Different methods were developed and applied in cells, 817,819,820 among which fast photochemical oxidation of proteins (FPOP) appears to be promising. 817,821,822 This technique generates chemical reactions on the microsecond timescale, which can reveal the solvent-accessible residues of thousands of proteins from about 10 million cells, [823][824][825][826] or from 10,000 Caenorhabditis elegans. 827,828 While FPOP in vitro has been shown to be capable of characterizing epitope mapping, conformational changes and even of yielding good protein structure predictions, 821,829 it is still in a development phase in cells.…”

Section: Mass-spectrometrymentioning

confidence: 99%

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: Mass-spectrometrymentioning

confidence: 99%

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

Theillet

2022

Chem. Rev.

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“…Current studies in this platform incubator have been performed on HEK293T cells and in C. elegans. The IC-FPOP method has been shown to be compatible with a wide variety of cell lines including Chinese hamster ovary (CHO), Vero, MCF-7, and MCF10-A cells 19 . Since the general IC-FPOP method is translatable to this static platform, these cell lines should be amenable for study using PIXY as well.…”

Section: Discussionmentioning

confidence: 99%

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins

Johnson

Punshon‐Smith

Espino

et al. 2021

JoVE

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Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO 2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H 2 O 2 concentration in

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“…Recent improvements in the IC-FPOP flow system enabled the analysis of single cells, , ensuring that cells are individually exposed to similar levels of radiation and avoiding cell clogging in the capillary. Moreover, the use of IC-FPOP was combined with 2D LC-MS/MS for the analysis of multiple eukaryotic cell lines, e.g., HEK 293T, CHO, MCF-10A, and MCF-7 . This advance in the method could be useful for future FPOP studies using different cells as models for human diseases.…”

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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Validation of the Applicability of In-Cell Fast Photochemical Oxidation of Proteins across Multiple Eukaryotic Cell Lines

Cited by 15 publications

References 46 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

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins

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

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