Analyzing the structure of macromolecules in their native cellular environment using hydroxyl radical footprinting

Chea, Emily E.; Jones, Lisa M.

doi:10.1039/c7an01323j

Cited by 21 publications

(19 citation statements)

References 54 publications

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“…Investigate solvent-exposed proteins in live cells [29]. The fast turnaround of the labeling can maintain cell viability while probing protein-solvent accessibility in a native environment [30].…”

Section: Fpop Applicationsmentioning

confidence: 99%

Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology

Zhang

Cheng

Rempel

et al. 2018

Methods

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Fast photochemical oxidation of proteins (FPOP) is a footprinting technique used in mass spectrometry-based structural proteomics. It has been applied to solve a variety of problems in different areas of biology. A FPOP platform requires a laser, optics, and sample flow path properly assembled to enable fast footprinting. Sample preparation, buffer conditions, and reagent concentrations are essential to obtain reasonable oxidations on proteins. FPOP samples can be analyzed by LC-MS methods to measure the modification extent, which is a function of the solvent-accessible surface area of the protein. The platform can be expanded to accommodate several new approaches, including dose-response studies, new footprinting reagents, and two-laser pump-probe experiments. Here, we briefly review FPOP applications and in a detailed manner describe the procedures to set up an FPOP protein footprinting platform.

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Section: Fpop Applicationsmentioning

confidence: 99%

Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology

Zhang

Cheng

Rempel

et al. 2018

Methods

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“…Each of these methods have been used for proteome wide structural biology to elucidate protein structure and ultimately function within the complex cellular environment. IC-FPOP is a HRPF technique that utilizes hydroxyl radicals to oxidatively modify solvent exposed side chains of amino acids, probing protein structure and protein-ligand interactions within viable cells 13 . IC-FPOP is an improvement to initial HRPF in live cells that used Fenton chemistry to generate radicals on the minutes timescale 14 .…”

Section: Discussionmentioning

confidence: 99%

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Chea

Rinas

Espino

et al. 2020

JoVE

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Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein's solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins. Video LinkThe video component of this article can be found at https://www.jove.com/video/60911/ Initial studies of IC-FPOP successfully probed 105 proteins ranging in protein abundance and cellular localization. To improve the IC-FPOP method, Rinas et al. developed a microflow system for single cell flow 9 . The enhancement of the original flow system limits cell aggregation and increases the available H 2 O 2 available for irradiation. In the initial flow system, cells clumping in the silica tubing resulted in clogs and uneven irradiation. The incorporation of two streams of a sheath buffer hydrodynamically focuses the cells, allowing them to flow individually past the laser. The incorporation of a separate syringe for the H 2 O 2 enables more controlled and optimizable exposure time allowing higher H 2 O 2 concentrations without adverse effects. Also, limiting the incubation time limits the breakdown of H 2 O 2 by endogenous catalase. By incorporating this new flow system, the detected number of proteins with an FPOP modification increased 13-fold, thus expanding the capabilities of this method to probe a multitude of proteins in living cells. In this protocol a general IC-FPOP experiment is described focusing on the assembly of the IC-FPOP flow system.

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“…The interaction of biomolecules (e.g., RNA) with MPs will also complement our understanding of MPs [102]. The potential of in-cell MS methods is also growing wherein significant advances are likely to be made in the years ahead, building on the studies of Jones and coworkers [103][104][105]. Rather than in vitro and in reconstituted systems, in-cell analysis enables MPs to be studied directly in cellular membranes.…”

Section: Summary and Perspectivesmentioning

confidence: 99%

Advances in mass spectrometry‐based footprinting of membrane proteins

Gross

2022

Proteomics

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Structural biology is entering an exciting time where many new high‐resolution structures of large complexes and membrane proteins (MPs) are determined regularly. These advances have been driven by over 15 years of technological improvements, first in macromolecular crystallography, and recently in cryo‐electron microscopy. Obtaining information about MP higher order structure and interactions is also a frontier, important but challenging owing to their unique properties and the need to choose suitable detergents/lipids for their study. The development of mass spectrometry (MS), both instruments and methodology in the past 10 years, has also advanced it as a complementary method to study MP structure and interactions. In this review, we discuss advances in MS‐based footprinting for MPs and highlight recent methodologies that offer new promise for MP study by chemical footprinting and mass spectrometry.

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Analyzing the structure of macromolecules in their native cellular environment using hydroxyl radical footprinting

Cited by 21 publications

References 54 publications

Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology

Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Advances in mass spectrometry‐based footprinting of membrane proteins

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