Fast Photochemical Oxidation of Proteins for Epitope Mapping

Jones, Lisa M.; Sperry, Justin B.; Carroll, James A.; Gross, Michael L.

doi:10.1021/ac2007366

Cited by 111 publications

(110 citation statements)

References 42 publications

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“…Hydrogen peroxide (H 2 O 2 ) (30%), formic acid, PBS, L-glutamine, and L-methionine were obtained from Sigma-Aldrich. The FPOP labeling protocol was modified from that previously described (52,53,(66)(67)(68)(69). The FPOP method uses a KrF excimer laser to generate 248-nm wavelength laser pulses to photolyze molecular H 2 O 2 into •OH radicals that covalently modify solvent-exposed side chains of amino acids (described in refs.…”

Section: Methodsmentioning

confidence: 99%

Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting

Poor¹,

Jones

Sood

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Significance The activities of the fusion proteins that mediate virus–cell fusion are an absolute requirement for virus entry and infectivity of enveloped viruses such as HIV, influenza virus, measles virus, and respiratory syncytia virus, among others. Viral fusion proteins are translated initially in a metastable prefusion state and, upon triggering, undergo an extensive and irreversible refolding process. Membrane fusion is coupled to the energy released by the fusion proteins adopting a stable, low-energy postfusion state. Here we use oxidative footprinting of the parainfluenza virus 5 fusion protein to reveal new details of this critical event in the viral lifecycle. A greater understanding of the dynamic nature of these metastable proteins may reveal novel opportunities for the development of targeted therapeutics.

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Section: Methodsmentioning

confidence: 99%

Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting

Poor¹,

Jones

Sood

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Two appropriate approaches are hydrogen-deuterium exchange (HDX) [30-36] and oxidative labeling (e.g., OH radicals, FPOP) [37-40]. These methods are sensitive, can accommodate large proteins, and can be used with the protein in native or near-native states.…”

Section: Introductionmentioning

confidence: 99%

Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

Yan

Chen

Wei

et al. 2014

J. Am. Soc. Mass Spectrom.

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Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the tenth type III domain of human fibronectin (10Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human EGFR, we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein-protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFRAddnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitope from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.

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“…Post-FPOP, the yeast lysate samples were subjected to a two-step digestion process as previously described [16]. Each sample was acetone precipitated [17] and resuspended in 8 M urea 150 mM tris-HCL pH 8.5 buffer.…”

Section: Proteolysismentioning

confidence: 99%

Fast Photochemical Oxidation of Proteins Coupled to Multidimensional Protein Identification Technology (MudPIT): Expanding Footprinting Strategies to Complex Systems

Rinas

Jones

2014

J. Am. Soc. Mass Spectrom.

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Peptides containing the oxidation products of hydroxyl radical-mediated protein footprinting experiments are typically much less abundant than their unoxidized counterparts. This is inherent to the design of the experiment as excessive oxidation may lead to undesired conformational changes or unfolding of the protein, skewing the results. Thus, as the complexity of the systems studied using this method expands, the detection and identification of these oxidized species can be increasingly difficult with the limitations of data-dependent acquisition (DDA) and one-dimensional chromatography. Here we report the application of multidimensional protein identification technology (MudPIT) in combination with hydroxyl radical footprinting as a method to increase the identification of quantifiable peptides in these experiments. Using this method led to a 37% increase in unique peptide identifications as well as a 70% increase in protein group identifications over one-dimensional data-dependent acquisition on the same samples. Furthermore, we demonstrate the combination of these methods as a means to investigate megadalton complexes.

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Fast Photochemical Oxidation of Proteins for Epitope Mapping

Cited by 111 publications

References 42 publications

Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting

Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting

Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

Fast Photochemical Oxidation of Proteins Coupled to Multidimensional Protein Identification Technology (MudPIT): Expanding Footprinting Strategies to Complex Systems

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