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Inline Liquid Chromatography-Fast Photochemical Oxidation of Proteins Allows for Targeted Structural Analysis of Conformationally Heterogeneous Mixtures

ABSTRACT: The size exclusion was used to separate the mixture of holomyoglobin and apomyoglobin as well as native and aggregated adalimumab, just before laser irrdiation for FPOP. The footprint of the individual proteins were determined.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Bos Taurus (bovine)

SUBMITTER: Sandeep Misra

LAB HEAD: Joshua S. Sharp

PROVIDER: PXD023402 | Pride | 2022-02-16

REPOSITORIES: pride

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			Action	DRS
	Adalimumab-aggregated-1.raw	Raw
	Adalimumab-aggregated-1.xlsx	Xlsx
	Adalimumab-aggregated-2.raw	Raw
	Adalimumab-aggregated-2.xlsx	Xlsx
	Adalimumab-aggregated-3.raw	Raw

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Inline Liquid Chromatography-Fast Photochemical Oxidation of Proteins for Targeted Structural Analysis of Conformationally Heterogeneous Mixtures.

Tadi Surendar S Misra Sandeep K SK Sharp Joshua S JS

Analytical chemistry 20210209 7

Structural analysis of proteins in a conformationally heterogeneous mixture has long been a difficult problem in structural biology. In structural analysis by covalent labeling mass spectrometry, conformational heterogeneity results in data reflecting a weighted average of all conformers, complicating data analysis and potentially causing misinterpretation of results. Here, we describe a method coupling size-exclusion chromatography (SEC) with hydroxyl radical protein footprinting using inline f ...[more]

PMID: 33560821

Similar Datasets

Inline Liquid Chromatography-Fast Photochemical Oxidation of Proteins for Targeted Structural Analysis of Conformationally Heterogeneous Mixtures.

Project description:Structural analysis of proteins in a conformationally heterogeneous mixture has long been a difficult problem in structural biology. In structural analysis by covalent labeling mass spectrometry, conformational heterogeneity results in data reflecting a weighted average of all conformers, complicating data analysis and potentially causing misinterpretation of results. Here, we describe a method coupling size-exclusion chromatography (SEC) with hydroxyl radical protein footprinting using inline fast photochemical oxidation of proteins (FPOP). Using a controlled synthetic mixture of holomyoglobin and apomyoglobin, we validate that we can achieve accurate footprints of each conformer using LC-FPOP when compared to offline FPOP of each pure conformer. We then applied LC-FPOP to analyze the adalimumab heat-shock aggregation process. We found that the LC-FPOP footprint of unaggregated adalimumab was consistent with a previously published footprint of the native IgG. The LC-FPOP footprint of the aggregation product indicated that heat-shock aggregation primarily protected the hinge region, suggesting that this region is involved with the heat-shock aggregation process of this molecule. LC-FPOP offers a new method to probe dynamic conformationally heterogeneous mixtures that can be separated by SEC such as biopharmaceutical aggregates and to obtain accurate information on the topography of each conformer.

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The Flash Oxidation (FOX) System: A Novel Laser-free Fast Photochemical Oxidation Protein Footprinting Platform

Project description:To validate the FOX flash photolysis system for hydroxyl radical footprinting of proteins experiments, TNF-alpha was oxidized in the presence and absence of a specific monoclonal antibody, adalimumab. The peptides that show decrease in oxidation correlate well with the known epitope for adalimumab.

2022-08-12 | PXD023169 | Pride

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

Project description: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 increased structural information. CPEs aid in the delivery of hydrogen peroxide inside C. elegans by disturbing the highly order 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.

2020-06-12 | PXD019290 | Pride

Data-Independent Acquisition Represents a Promising Alternative for Fast Photochemical Oxidation of Proteins (FPOP) Samples Analysis

Project description:We propose a novel approach for FPOP data analysis, utilizing DIA data. The HbHp protein complex was analyzed by FPOP and measured on timsToF SCP in DIA, DDA and MS modes. The IDs of modified peptides were quantified for each acquisition mode and the extent of modification was calculated on the level of peptides. The reproducibility was evaluated by coefficients of variation.This work was mainly financially supported by the Czech Science Foundation (22-27695S), the Technology Agency of the Czech Republic (ODEEP-EU TH86010001), the Ministry of Education, Youth and Sports of the Czech Republic grant PHOTOMACHINES - Photosynthetic cell redesign for high yields of therapeutic peptides (CZ.02.01.01/00/22_008/0004624) and the Academy of Sciences of the Czech Republic (RVO: 61388971).

2024-07-08 | PXD050132 | Pride

Triphilic Ionic-Liquid Mixtures: Fluorinated and Non-fluorinated Aprotic Ionic-Liquid Mixtures.

Project description:We present here the possibility of forming triphilic mixtures from alkyl- and fluoroalkylimidazolium ionic liquids, thus, macroscopically homogeneous mixtures for which instead of the often observed two domains-polar and nonpolar-three stable microphases are present: polar, lipophilic, and fluorous ones. The fluorinated side chains of the cations indeed self-associate and form domains that are segregated from those of the polar and alkyl domains. To enable miscibility, despite the generally preferred macroscopic separation between fluorous and alkyl moieties, the importance of strong hydrogen bonding is shown. As the long-range structure in the alkyl and fluoroalkyl domains is dependent on the composition of the liquid, we propose that the heterogeneous, triphilic structure can be easily tuned by the molar ratio of the components. We believe that further development may allow the design of switchable, smart liquids that change their properties in a predictable way according to their composition or even their environment.

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Diffusion in liquid mixtures.

Project description:The understanding of transport and mixing in fluids in the presence and in the absence of external fields and reactions represents a challenging topic of strategic relevance for space exploration. Indeed, mixing and transport of components in a fluid are especially important during long-term space missions where fuels, food and other materials, needed for the sustainability of long space travels, must be processed under microgravity conditions. So far, the processes of transport and mixing have been investigated mainly at the macroscopic and microscopic scale. Their investigation at the mesoscopic scale is becoming increasingly important for the understanding of mass transfer in confined systems, such as porous media, biological systems and microfluidic systems. Microgravity conditions will provide the opportunity to analyze the effect of external fields and reactions on optimizing mixing and transport in the absence of the convective flows induced by buoyancy on Earth. This would be of great practical applicative relevance to handle complex fluids under microgravity conditions for the processing of materials in space.

| S-EPMC9842720 | biostudies-literature

Homopolymer micelles in heterogeneous solvent mixtures.

Project description:Amphiphilic homopolymers containing a hydrophilic and a hydrophobic functionality in each monomer unit have been shown to form polar or apolar containers depending on the solvent environment. When presented with a mixture of solvents, these polymeric containers are capable of releasing certain guest molecules. The fundamental mechanism behind these properties is investigated, and the utility of these assemblies in separations has been demonstrated with an example.

| S-EPMC2532981 | biostudies-literature

Fast photochemical oxidation of proteins for epitope mapping.

Project description:The growing use of monoclonal antibodies as therapeutics underscores the importance of epitope mapping as an essential step in characterizing antibody-antigen complexes. The use of protein footprinting coupled with mass spectrometry, which is emerging as a tool in structural biology, offers opportunities to map antibody-binding regions of antigens. We report here the use of footprinting via fast photochemical oxidation of proteins (FPOP) with OH radicals to characterize the epitope of the serine protease thrombin. The data correlate well with previously published results that determined the epitope of thrombin. This study marks the first time oxidative labeling has been used for epitope mapping.

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Fast Photochemical Oxidation of Proteins Coupled with Mass Spectrometry.

Project description:BackgroundDetermination of the composition and some structural features of macromolecules can be achieved by using structural proteomics approaches coupled with mass spectrometry (MS). One approach is hydroxyl radical protein footprinting whereby amino-acid side chains are modified with reactive reagents to modify irreversibly a protein side chain. The outcomes, when deciphered with mass-spectrometry-based proteomics, can increase our knowledge of structure, assembly, and conformational dynamics of macromolecules in solution. Generating the hydroxyl radicals by laser irradiation, Hambly and Gross developed the approach of Fast Photochemical Oxidation of Proteins (FPOP), which labels proteins on the sub millisecond time scale and provides, with MS analysis, deeper understanding of protein structure and protein-ligand and protein- protein interactions. This review highlights the fundamentals of FPOP and provides descriptions of hydroxyl-radical and other radical and carbene generation, of the hydroxyl labeling of proteins, and of determination of protein modification sites. We also summarize some recent applications of FPOP coupled with MS in protein footprinting.ConclusionWe survey results that show the capability of FPOP for qualitatively measuring protein solvent accessibility on the residue level. To make these approaches more valuable, we describe recent method developments that increase FPOP's quantitative capacity and increase the spatial protein sequence coverage. To improve FPOP further, several new labeling reagents including carbenes and other radicals have been developed. These growing improvements will allow oxidative- footprinting methods coupled with MS to play an increasingly significant role in determining the structure and dynamics of macromolecules and their assemblies.

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A physicochemical investigation of ionic liquid mixtures.

Project description:Ionic liquids have earned the reputation of being 'designer solvents' due to the wide range of accessible properties and the degree of fine-tuning afforded by varying the constituent ions. Mixtures of ionic liquids offer the opportunity for further fine-tuning of properties. A broad selection of common ionic liquid cations and anions are employed to create a sample of binary and reciprocal binary ionic liquid mixtures, which are analysed and described in this paper. Physical properties such as the conductivity, viscosity, density and phase behaviour (glass transition temperatures) are examined. In addition, thermal stabilities of the mixtures are evaluated. The physical properties examined for these formulations are found to generally adhere remarkably closely to ideal mixing laws, with a few consistent exceptions, allowing for the facile prediction and control of properties of ionic liquid mixtures.

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