Publications on FOX™ HRPF technology

Interested in learning more about hydroxyl radical protein foot-printing? Check out the following publications that have been authored by members of our illustrious GenNext Team.

Misra S.K., Orlando R., Weinberger S.R. and Sharp J.S. (2019) Compensated Hydroxyl Radical Protein Footprinting Measures Buffer and Excipient Effects on Conformation and Aggregation in an Adalimumab Biosimilar. AAPS J 21, 8


Unlike small molecule drugs, therapeutic proteins must maintain the proper higher-order structure (HOS) in order to maintain safety and efficacy. Due to the sensitivity of many protein systems, even small changes due to differences in protein expression or formulation can alter HOS. Previous work has demonstrated how hydroxyl radical protein footprinting (HRPF) can sensitively detect changes in protein HOS by measuring the average topography of the protein monomers, as well as identify specific regions of the therapeutic protein impacted by the conformational changes. However, HRPF is very sensitive to the radical scavenging capacity of the buffer; addition of organic buffers and/or excipients can dramatically alter the HRPF footprint without affecting protein HOS. By compensating for the radical scavenging effects of different adalimumab biosimilar formulations using real-time adenine dosimetry, we identify that sodium citrate buffer causes a modest decrease in average solvent accessibility compared to sodium phosphate buffer at the same pH. We find that the addition of polysorbate 80 does not alter the conformation of the biosimilar in either buffer, but it does provide substantial protection from protein conformational perturbation during short periods of exposure to high temperature. Compensated HRPF measurements are validated and contextualized by dynamic light scattering (DLS), which suggests that changes in adalimumab biosimilar aggregation are major drivers in measured changes in protein topography. Overall, compensated HRPF accurately measured conformational changes in adalimumab biosimilar that occurred during formulation changes and identified the effect of formulation changes on protection of HOS from temperature extremes.

Abolhasani Khaje N. and Sharp J.S. (2019) Rapid Quantification of Peptide Oxidation Isomers from Complex Mixtures. Anal Chem 92, 3834-3843


Hydroxyl radical protein footprinting (HRPF) is a powerful technique for probing changes in protein topography, based on quantifying the amount of oxidation of different regions of a protein. While quantification of HRPF oxidation at the peptide level is relatively common and straightforward, quantification at the residue level is challenging because of the influence of oxidation on MS/MS fragmentation and the large number of complex and only partially chromatographically resolved isomeric peptide oxidation products. HRPF quantification of isomeric peptide oxidation products (where the peptide sequence is the same but isomeric oxidation products are formed at different sites) at the residue level by electron transfer dissociation tandem mass spectrometry (ETD MS/MS) has been demonstrated in both model peptides and HRPF products, but the method is hampered by the partial separation of oxidation isomers by reversed phase chromatography. This requires custom MS/MS methods to equally sample all isomeric oxidation products across their elution window, greatly increasing method development time and reducing the oxidation products quantified in a single LC-MS/MS run. Here, we present a zwitterionic hydrophilic interaction capillary chromatography (ZIC-HILIC) method to ideally coelute all isomeric peptide oxidation products while separating different peptides. This allows us to relatively quantify peptide oxidation isomers using an ETD MS/MS spectrum acquired at any point across the single peptide oxidation isomer peak, greatly simplifying data acquisition and data analysis.

Roush A.E., Riaz M., Misra S.K., Weinberger S.R. and Sharp J.S. (2019) Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(Hydroxymethyl)Aminomethane. J Am Soc Mass Spectrom 31, 169-172


Fast photochemical oxidation of proteins (FPOP) is a powerful covalent labeling tool that uses hydroxyl radicals generated by laser flash photolysis of hydrogen peroxide to footprint protein surfaces. Because radical production varies with many experimental parameters, hydroxyl radical dosimeters have been introduced to track the effective radical dosage experienced by the protein analyte. FPOP experiments performed using adenine optical radical dosimetry containing protein in Tris buffer demonstrated unusual dosimetry behavior. We have investigated the behavior of Tris under oxidative conditions in detail. We find that Tris can act as a novel gain-of-signal optical hydroxyl radical dosimeter in FPOP experiments. This new dosimeter is also amenable to inline real-time monitoring, thereby allowing real-time adjustments to compensate for differences in samples for their quenching ability.

Tadi S. and Sharp J.S. (2019) Top-Down ETD-MS Provides Unreliable Quantitation of Methionine Oxidation. J Biomol Tech 30, 50-57 (Received the Journal of Biomolecular Techniques Outstanding Manuscript Award for 2019.)


Methionine oxidation plays a critical role in many processes of biologic and biomedical importance, including cellular redox responses and stability of protein pharmaceuticals. Bottom-up methods for analysis of methionine oxidation can suffer from incomplete sequence coverage, as well as an inability to readily detect correlated oxidation between 2 or more methionines. However, the methodology for quantifying protein oxidation in top-down analyses is lacking. Previous work has shown that electron transfer dissociation (ETD)–based tandem mass spectrometry (MS/MS) fragmentation offers accurate and precise quantification of amino acid oxidation in peptides, even in complex samples. However, the ability of ETD-based MS/MS fragmentation to accurately quantify amino acid oxidation of proteins in a top-down manner has not been reported. Using apomyoglobin and calmodulin as model proteins, we partially converted methionines into methionine sulfoxide by incubation in H2O2. Using top-down ETD-based fragmentation, we quantified the amount of oxidation of various ETD product ions and compared the quantified values with those from traditional bottom-up analysis. We find that overall quantification of methionine oxidation by top-down MS/MS ranges from good agreement with traditional bottom-up methods to vast differences between the 2 techniques, including missing oxidized product ions and large differences in measured oxidation quantities. Care must be taken in transitioning ETD-based quantitation of oxidation from the peptide level to the intact protein level.

Sharp, J.S., J.M. Becker, and R.L. Hettich, Protein surface mapping by chemical oxidation: structural analysis by mass spectrometry. Anal Biochem, 2003. 313(2): p. 216-25.


The solvent-accessible surface area of proteins is important in biological function for many reasons, including protein-protein interactions, protein folding, and catalytic sites. Here we present a chemical technique to oxidize amino acid side chains in a model protein, apomyoglobin, and subsequent elucidation of the effect of solvent accessibility on the sites of oxidation. Under conditions of low protein oxidation (zero to three oxygen atoms added per apomyoglobin molecule), we have positively identified five oxidation sites by liquid chromatography-tandem mass spectrometry and high-resolution Fourier transform mass spectrometry. Our results indicate that all oxidized amino acids, with the exception of methionine, have highly solvent-accessible side chains, but the rate of oxidation may not be dictated solely by solvent accessibility and amino acid identity.

Sharp, J.S., J.M. Becker, and R.L. Hettich, Analysis of protein solvent accessible surfaces by photochemical oxidation and mass spectrometry. Anal Chem, 2004. 76(3): p. 672-83.


Protein surfaces are important in most biological processes, including proteinprotein interactions, enzymatic catalysis, and protein-ligand binding. We report a method in which hydroxyl radicals generated by a rapid-UV irradiation of a 15% hydrogen peroxide solution were utilized to oxidize specific amino acid side chains of two model proteins (lysozyme, beta-lactoglobulin A), according to the residues’ chemical reactivities and the solvent accessibility of the reactive carbons and sulfurs in the residue. Oxidized peptides generated by tryptic digestion were identified by electrospray-Fourier transform mass spectrometry. The specific sites of the stable modification were then identified by reverse-phase liquid chromatography coupled to quadropole ion trap tandem mass spectrometry. The solvent accessibility of the residue was shown to directly affect the rate of oxidation by this method (with the exception of methionine), supporting its use as a rapid measure of the solvent accessibility of specific residues, and in some cases, individual atoms.

harp, J.S., et al., Photochemical surface mapping of C14S-Sml1p for constrained computational modeling of protein structure. Anal Biochem, 2005. 340(2): p. 201-212.


Photochemically generated hydroxyl radicals were used to map solvent-exposed regions in the C14S mutant of the protein Sml1p, a regulator of the ribonuclease reductase enzyme Rnr1p in Saccharomyces cerevisiae. By using high-performance mass spectrometry to characterize the oxidized peptides created by the hydroxyl radical reactions, amino acid solvent-accessibility data for native and denatured C14S Sml1p that revealed a solvent-excluding tertiary structure in the native state were obtained. The data on solvent accessibilities of various amino acids within the protein were then utilized to evaluate the de novo computational models generated by the HMMSTR/Rosetta server. The top five models initially generated by the server all disagreed with both published nuclear magnetic resonance (NMR) data and the solvent-accessibility data obtained in this study. A structural model adjusted to fit the previously reported NMR data satisfied most of the solvent-accessibility constraints. Through minor adjustment of the rotamers of two amino acid side chains for this latter structure, a model that not only provided a lower energy conformation but also completely satisfied previously reported data from NMR and tryptophan fluorescence measurements, in addition to the solvent-accessibility data presented here, was generated.

Sharp, J.S., et al., Structural characterization of the E2 glycoprotein from Sindbis by lysine biotinylation and LC-MS/MS. Virology, 2006. 348(1): p. 216-23.


Sindbis is an Alphavirus capable of infecting and replicating in both vertebrate and invertebrate hosts. Mature Sindbis virus particles consist of an inner capsid surrounded by a host-derived lipid bilayer, which in turn is surrounded by a protein shell consisting of the E1 and E2 glycoproteins. While a homolog of the E1 glycoprotein has been structurally characterized, the amount of structural data on the E2 glycoprotein is considerably less. In this study, the organization of the E2 glycoprotein was probed by surface biotinylation of intact virions. The virus remained fully infectious, demonstrating that the biotinylation did not alter the topology of the proteins involved in infection. Seven sites of modification were identified in the E2 glycoprotein (K70, K76, K97, K131, K149, K202, and K235), while one site of modification in the E1 glycoprotein (K16) was identified, confirming that the E1 protein is almost completely buried in the virus structure.

5. Watson, C. and J.S. Sharp, Conformational Analysis of Therapeutic Proteins by Hydroxyl Radical Protein Footprinting. AAPS J, 2012. 14(2): p. 206-217.


Unlike small molecule drugs, therapeutic protein pharmaceuticals must not only have the correct amino acid sequence and modifications, but also the correct conformation to ensure safety and efficacy. Here, we describe a method for comparison of therapeutic protein conformations by hydroxyl radical protein footprinting using liquid chromatography-mass spectrometry (LC-MS) as an analytical platform. Hydroxyl radical protein footprinting allows for rapid analysis of the conformation of therapeutic proteins based on the apparent rate of oxidation of various amino acids by hydroxyl radicals generated in situ. Conformations of Neupogen®, a patented granulocyte colony-stimulating factor (GCSF), were compared to several expired samples of recombinant GCSF, as well as heat-treated Neupogen®. Conformations of different samples of the therapeutic proteins interferon α-2A and erythropoietin were also compared. Differences in the hydroxyl radical footprint were measured between Neupogen® and the expired or mishandled GCSF samples, and confirmed by circular dichroism spectroscopy. Samples that had identical circular dichroism spectra were also found to be indistinguishable by hydroxyl radical footprinting. The method is applicable to a wide variety of therapeutic proteins and formulations through the use of separations techniques to clean up the protein samples after radical oxidation. The reaction products are stable, allowing for flexibility in sample handling, as well as archiving and reanalysis of samples. Initial screening can be performed on small amounts of therapeutic protein with minimal training in LC-MS, but samples with structural differences from the reference can be more carefully analyzed by LC-MS/MS to attain higher spatial resolution, which can aid in engineering and troubleshooting.

Li, Z., et al., High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface. Journal of Biological Chemistry, 2015. 290(17): p. 10729-10740.


Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes.

Li, X., et al., Structural Analysis of the Glycosylated Intact HIV-1 gp120–b12 Antibody Complex Using Hydroxyl Radical Protein Footprinting. Biochemistry, 2017. 56(7): p. 957-970.


Glycoprotein gp120 is a surface antigen and virulence factor of human immunodeficiency virus 1. Broadly neutralizing antibodies (bNAbs) that react to gp120 from a variety of HIV isolates offer hope for the development of broadly effective immunogens for vaccination purposes, if the interactions between gp120 and bNAbs can be understood. From a structural perspective, gp120 is a particularly difficult system because of its size, the presence of multiple flexible regions, and the large amount of glycosylation, all of which are important in gp120-bNAb interactions. Here, the interaction of full-length, glycosylated gp120 with bNAb b12 is probed using high-resolution hydroxyl radical protein footprinting (HR-HRPF) by fast photochemical oxidation of proteins. HR-HRPF allows for the measurement of changes in the average solvent accessible surface area of multiple amino acids without the need for measures that might alter the protein conformation, such as mutagenesis. HR-HRPF of the gp120-b12 complex coupled with computational modeling shows a novel extensive interaction of the V1/V2 domain, probably with the light chain of b12. Our data also reveal HR-HRPF protection in the C3 domain caused by interaction of the N330 glycan with the b12 light chain. In addition to providing information about the interactions of full-length, glycosylated gp120 with b12, this work serves as a template for the structural interrogation of full-length glycosylated gp120 with other bNAbs to better characterize the interactions that drive the broad specificity of the bNAb.

Sharp, J.S., FPOP HRPF for small molecule target binding site elucidation, 2017, University of Mississippi

Xie, B., et al., Quantitative Protein Topography Measurements by High Resolution Hydroxyl Radical Protein Footprinting Enable Accurate Molecular Model Selection. Scientific reports, 2017. 7(1): p. 4552.


We report an integrated workflow that allows mass spectrometry-based highresolution hydroxyl radical protein footprinting (HR-HRPF) measurements to accurately measure the absolute average solvent accessible surface area () of amino acid side chains. This approach is based on application of multi-point HR-HRPF, electron-transfer dissociation (ETD) tandem MS (MS/MS) acquisition, measurement of effective radical doses by radical dosimetry, and proper normalization of the inherent reactivity of the amino acids. The accuracy of the resulting measurements was tested by using well-characterized protein models. Moreover, we demonstrated the ability to use measurements from HR-HRPF to differentiate molecular models of high accuracy (<3 Å backbone RMSD) from models of lower accuracy (>4 Å backbone RMSD). The ability of data from HR-HRPF to differentiate molecular model quality was found to be comparable to that of data obtained from X-ray crystal structures, indicating the accuracy and utility of HR-HRPF for evaluating the accuracy of computational models.

Sharp, J.S., et al., Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry. Anal Chem, 2018.


Hydroxyl radical protein footprinting (HRPF) is a powerful method for measuring protein topography, allowing researchers to monitor events that alter the solvent accessible surface of a protein (e.g., ligand binding, aggregation, conformational changes, etc.) by measuring changes in the apparent rate of reaction of portions of the protein to hydroxyl radicals diffusing in solution. Fast Photochemical Oxidation of Proteins (FPOP) offers an ultrafast benchtop method for radical generation for HRPF, photolyzing hydrogen peroxide using a UV laser to generate high concentrations of hydroxyl radicals that are consumed on roughly a microsecond time scale. The broad reactivity of hydroxyl radicals means that almost anything added to the solution (e.g., ligands, buffers, excipients, etc.) will scavenge hydroxyl radicals, altering their half-life and changing the effective radical concentration experienced by the protein. Similarly, minute changes in peroxide concentration, laser fluence, and buffer composition can alter the effective radical concentration, making reproduction of data challenging. Here, we present a simple method for radical dosimetry that can be carried out as part of the FPOP workflow, allowing for measurement of effective radical concentration in real time. Additionally, by modulating the amount of radical generated, we demonstrate that effective hydroxyl radical yields in FPOP HRPF experiments carried out in buffers with widely differing levels of hydroxyl radical scavenging capacity can be compensated on the fly, yielding statistically indistinguishable results for the same conformer. This method represents a major step in transforming FPOP into a robust and reproducible technology capable of probing protein structure in a wide variety of contexts.

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