Hydroxyl Radical Protein Footprinting Research Articles

Epitope mapping of SARS-CoV-2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls

Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogen‑deuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups.This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis.Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD.The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.

Evaluating Mass Spectrometry-Based Hydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation System against a Synchrotron X-ray Beamline.

Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation (XFP) and mass spectrometry is a well-validated structural biology method that provides critical insights into macromolecular structural dynamics, such as determining binding sites, measuring affinity, and mapping epitopes. Numerous alternative sources for generating the hydroxyl radicals (•OH) needed for HRPF, such as laser photolysis and plasma irradiation, complement synchrotron-based HRPF, and a recently developed commercially available instrument based on flash lamp photolysis, the FOX system, enables access to laboratory benchtop HRPF. Here, we evaluate performing HRPF experiments in-house with a benchtop FOX instrument compared to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase (LOx) as a model system, we carried out •OH labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed under high glucose concentrations to mimic the highly scavenging conditions present in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the FOX and XFP HRPF methods was compared, and we found that tuning the •OH dosage enabled optimal labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of FOX and XFP labeling approaches, demonstrating that benchtop instruments such as the FOX photolysis system can increase both the throughput and the accessibility of the HRPF technique.

Dimethylthiourea as a Quencher in Hydroxyl Radical Protein Footprinting Experiments.

Hydroxyl radical protein footprinting (HRPF) is a mass-spectrometry-based method for studying protein structures, interactions, conformations, and folding. This method is based on the irreversible labeling of solvent-exposed amino acid side chains by hydroxyl radicals. While catalase is commonly used as a quencher after the labeling of a protein by the hydroxyl radicals to efficiently remove the remaining hydrogen peroxide, it has some disadvantages. Catalase quenching adds a relatively high amount of protein to the sample, limiting the sensitivity of the method due to dynamic range issues and causing significant issues when dealing with more complex samples. We evaluated dimethylthiourea (DMTU) as a replacement for catalase in the quenching HRPF reactions. We observed that DMTU is highly effective at quenching HRPF oxidation. DMTU does not cause the background protein issues that catalase does, resulting in an increased number of protein identifications from complex mixtures. We recommend the replacement of catalase quenching with DMTU for all HRPF experiments.

Unlatching of the stem domains in the Staphylococcus aureus pore-forming leukocidin LukAB influences toxin oligomerization

Staphylococcus aureus (S.aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S.aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric β-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here, we show that hydrogen voltage-gated channel 1 is also required for the cytotoxicity of all major LukAB variants. We demonstrate that while each receptor is sufficient to recruit LukAB to the plasma membrane, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contributes to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow LukAB to maintain cytotoxicity without CD11b. We discovered 30 mutations primarily localized in the stem domains of LukA and LukB that enable LukAB to exhibit full cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting, we show these mutations increase the solvent accessibility of the stem domain, priming LukAB for oligomerization. Together, our data support a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization.

Intact mass spectrometry screening to optimize hydroxyl radical dose for protein footprinting

Hydroxyl radical protein footprinting (HRPF) using synchrotron radiation is a well-validated method to assess protein structure in the native solution state. In this method, X-ray radiolysis of water generates hydroxyl radicals that can react with solvent accessible side chains of proteins, with mass spectrometry used to detect the resulting labeled products. An ideal footprinting dose provides sufficient labeling to measure the structure but not so much as to influence the results. The optimization of hydroxyl radical dose is typically performed using an indirect Alexa488 fluorescence assay sensitive to hydroxyl radical concentration, but full evaluation of the experiment's outcome relies upon bottom-up liquid chromatography mass spectrometry (LC-MS) measurements to directly determine sites and extent of oxidative labeling at the peptide and protein level. A direct evaluation of the extent of labeling to provide direct and absolute measurements of dose and “safe” dose ranges in terms of, for example, average numbers of labels per protein, would provide immediate feedback on experimental outcomes prior to embarking on detailed LC-MS analyses. To this end, we describe an approach to integrate intact MS screening of labeled samples immediately following exposure, along with metrics to quantify the extent of observed labeling from the intact mass spectra. Intact MS results on the model protein lysozyme were evaluated in the context of Alexa488 assay results and a bottom-up LC-MS analysis of the same samples. This approach provides a basis for placing delivered hydroxyl radical dose metrics on firmer technical grounds for synchrotron X-ray footprinting of proteins, with explicit parameters to increase the likelihood of a productive experimental outcome. Further, the method directs approaches to provide absolute and direct dosimetry for all types of labeling for protein footprinting.

Structural Investigation of Therapeutic Antibodies Using Hydroxyl Radical Protein Footprinting Methods.

Commercial monoclonal antibodies are growing and important components of modern therapies against a multitude of human diseases. Well-known high-resolution structural methods such as protein crystallography are often used to characterize antibody structures and to determine paratope and/or epitope binding regions in order to refine antibody design. However, many standard structural techniques require specialized sample preparation that may perturb antibody structure or require high concentrations or other conditions that are far from the conditions conducive to the accurate determination of antigen binding or kinetics. We describe here in this minireview the relatively new method of hydroxyl radical protein footprinting, a solution-state method that can provide structural and kinetic information on antibodies or antibody-antigen interactions useful for therapeutic antibody design. We provide a brief history of hydroxyl radical footprinting, examples of current implementations, and recent advances in throughput and accessibility.

Incorporation of D2O-Induced Fluorine Chemical Shift Perturbations into Ensemble-Structure Characterization of the ERalpha Disordered Region.

Structural characterization of intrinsically disordered proteins (IDPs) requires a concerted effort between experiments and computations by accounting for their conformational heterogeneity. Given the diversity of experimental tools providing local and global structural information, constructing an experimental restraint-satisfying structural ensemble remains challenging. Here, we use the disordered N-terminal domain (NTD) of the estrogen receptor alpha (ERalpha) as a model system to combine existing small-angle X-ray scattering (SAXS) and hydroxyl radical protein footprinting (HRPF) data and newly acquired solvent accessibility data via D2O-induced fluorine chemical shifting (DFCS) measurements. A new set of DFCS data for the solvent exposure of a set of 12 amino acid positions were added to complement previously acquired HRPF measurements for the solvent exposure of the other 16 nonoverlapping amino acids, thereby improving the NTD ensemble characterization considerably. We also found that while choosing an initial ensemble of structures generated from a different atomic-level force field or sampling/modeling method can lead to distinct contact maps even when the same sets of experimental measurements were used for ensemble-fitting, comparative analyses from these initial ensembles reveal commonly recurring structural features in their ensemble-averaged contact map. Specifically, nonlocal or long-range transient interactions were found consistently between the N-terminal segments and the central region, sufficient to mediate the conformational ensemble and regulate how the NTD interacts with its coactivator proteins.

Multiplex Chemical Labeling of Amino Acids for Protein Footprinting Structure Assessment.

Protein footprinting with mass spectrometry is an established structural biology technique for mapping solvent accessibility and assessing molecular-level interactions of proteins. In hydroxyl radical protein footprinting (HRPF), hydroxyl (OH) radicals generated by water radiolysis or other methods covalently label protein side chains. Because of the wide dynamic range of OH reactivity, not all side chains are easily detected in a single experiment. Novel reagent development and the use of radical chain reactions for labeling, including trifluoromethyl radicals, is a potential approach to normalize the labeling across a diverse set of residues. HRPF in the presence of a trifluoromethylation reagent under the right conditions could provide a "one-pot" reaction for multiplex labeling of protein side chains. Toward this goal, we have systematically evaluated amino acid labeling with the recently investigated Langlois' reagent (LR) activated by X-ray-mediated water radiolysis, followed by three different mass spectrometry methods. We compared the reactivity of CF3 and OH radical labeling for all 20 protein side chains in a competition-free environment. We found that all 20 amino acids exhibited CF3 or OH labeling in LR. Our investigations provide the evidence and knowledge set to perfect hydroxyl radical-activated trifluoromethyl chemistry as "one-pot" reaction for multiplex labeling of protein side chains to achieve higher resolution in HRPF.

Hydroxyl radical protein footprinting for analysis of higher order structure

Tabled 1aAdapted from Johnson et al. [11].Hydrogen deuterium exchangeGlycine ethyl esterDiethylpyrocarbonateN-Ethylmaleimide (NEM)Fast photochemical oxidation of proteinsLabeling timescaleSeconds to hoursSeconds to minutesMinutesMinutes to hoursMicrosecondsType of labelReversibleReversibleReversible for most residuesIrreversible for LysIrreversibleIrreversibleSensitivity/specificityBackbone amide groupsCarboxylic acidsNucleophilic residuesN terminus, His, Lys, Tyr, Ser, Thr, and CysCys19 out of 20 amino acids (excluding Gly)Chemicals/materialsCommonly used buffers under physiological conditionsQuench solutionspH 2.5 (strong or weak acid)0°CDenaturing (urea,guanidine HCl)Reducing agents (TCEP)Pepsin digestionEDC stock solutionprepared freshly in PBS bufferMES/NaOH (pH = 5.5),PBS (pH = 6, 6.5, 7, 7.5),HEPES (pH = 8, 8.5)DEPC in acetonitrile in a molar excess of 4 10 mM imidazole added to quench the reactionNEM solution10 mm HEPES5% DMSOVarious buffer solutions based on proteinH2O2free glutamine radical scavengerQuench solution: free methionine and catalase (in vitro)LimitationsReversibility of the label limits its use for complex samples that require longer separation timesCarboxylic acid-specific labeling limits the proteins that can be studiedFree cysteine scramblingCysteine-specific labeling limits the proteins that can be studiedResidue-reactivity differences, complex data analysis, and expense of excimer lasera Adapted from Johnson et al. [11]. Open table in a new tab Hydroxyl radicals can be generated using various methods, including Fenton chemistry, radiolysis of water, and photolysis of hydrogen peroxide. The labeling timescale is based on the radical generation method with some labeling proteins on the nano- or microsecond timescale, which is faster than all other footprinting methods. HRPF has been successful in studying HOS in varied applications, including epitope mapping, ion channel dynamics, and viral protein studies. HRPF has also been used to study intact cells and in vivo systems.ADVANTAGES:General labeling strategy that can modify 19 of the 20 amino acids.Labeling is compatible with several widely used buffer systems.The irreversible nature of the label provides flexibility for postlabeling sample handling.Can identify protein interaction sites and regions of conformational changes.Can analyze complex systems such as cell lysates, intact cells, and Caenorhabditis elegans.Advancements in dosimetry have increased the quantitative power of the method.There have been efforts to automate labeling leading to higher throughput.CHALLENGES:Data analysis is complex, time consuming, and there is not a universal data analysis platform.Generating hydroxyl radicals on a fast timescale requires special instrumentation.Lower resolution than other structural methods.Difficult to identify interacting proteins in complex systems.Declaration of interestsL.M.J. discloses a significant financial interest in GenNext Technologies, Inc., an early-stage company seeking to commercialize technologies for protein higher-order structure analysis. Hydroxyl radicals can be generated using various methods, including Fenton chemistry, radiolysis of water, and photolysis of hydrogen peroxide. The labeling timescale is based on the radical generation method with some labeling proteins on the nano- or microsecond timescale, which is faster than all other footprinting methods. HRPF has been successful in studying HOS in varied applications, including epitope mapping, ion channel dynamics, and viral protein studies. HRPF has also been used to study intact cells and in vivo systems. ADVANTAGES:General labeling strategy that can modify 19 of the 20 amino acids.Labeling is compatible with several widely used buffer systems.The irreversible nature of the label provides flexibility for postlabeling sample handling.Can identify protein interaction sites and regions of conformational changes.Can analyze complex systems such as cell lysates, intact cells, and Caenorhabditis elegans.Advancements in dosimetry have increased the quantitative power of the method.There have been efforts to automate labeling leading to higher throughput. General labeling strategy that can modify 19 of the 20 amino acids. Labeling is compatible with several widely used buffer systems. The irreversible nature of the label provides flexibility for postlabeling sample handling. Can identify protein interaction sites and regions of conformational changes. Can analyze complex systems such as cell lysates, intact cells, and Caenorhabditis elegans. Advancements in dosimetry have increased the quantitative power of the method. There have been efforts to automate labeling leading to higher throughput. CHALLENGES:Data analysis is complex, time consuming, and there is not a universal data analysis platform.Generating hydroxyl radicals on a fast timescale requires special instrumentation.Lower resolution than other structural methods.Difficult to identify interacting proteins in complex systems. Data analysis is complex, time consuming, and there is not a universal data analysis platform. Generating hydroxyl radicals on a fast timescale requires special instrumentation. Lower resolution than other structural methods. Difficult to identify interacting proteins in complex systems. Declaration of interestsL.M.J. discloses a significant financial interest in GenNext Technologies, Inc., an early-stage company seeking to commercialize technologies for protein higher-order structure analysis. L.M.J. discloses a significant financial interest in GenNext Technologies, Inc., an early-stage company seeking to commercialize technologies for protein higher-order structure analysis.

Validated determination of NRG1 Ig-like domain structure by mass spectrometry coupled with computational modeling

High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure. Independent determination of the protein structure by both HR-HRPF-based modeling and heteronuclear NMR was carried out, with results compared only after both processes were complete. The HR-HRPF-based model was highly similar to the lowest energy NMR model, with a backbone RMSD of 1.6 Å. To our knowledge, this is the first use of HR-HRPF-based modeling to determine a previously uncharacterized protein structure.

Laser-free flash oxidation (Fox) hydroxyl radical protein footprinting system accurately maps the paratope and epitope of TNFα bound to adalimumab

The Fox Protein Footprinting System is a novel hydroxyl radical protein footprinting (HRPF) method that uses a proprietary flash oxidation lamp to photolyze hydrogen peroxide to form hydroxyl radicals (⋅OH). The ⋅OH irreversibly modify solvent exposed side chains of amino acids. As solvent accessibility changes, the ⋅OH modification concordantly changes. With the addition of real time radical dosimetry, the effective ·OH concentration can be determined and easily adjusted, allowing conditions with varying scavenging effects to be easily compared.

Protein Structure Prediction with Mass Spectrometry Data.

Knowledge of protein structure is crucial to our understanding of biological function and is routinely used in drug discovery. High-resolution techniques to determine the three-dimensional atomic coordinates of proteins are available. However, such methods are frequently limited by experimental challenges such as sample quantity, target size, and efficiency. Structural mass spectrometry (MS) is a technique in which structural features of proteins are elucidated quickly and relatively easily. Computational techniques that convert sparse MS data into protein models that demonstrate agreement with the data are needed. This review features cutting-edge computational methods that predict protein structure from MS data such as chemical cross-linking, hydrogen-deuterium exchange, hydroxyl radical protein footprinting, limited proteolysis, ion mobility, and surface-induced dissociation. Additionally, we address future directions for protein structure prediction with sparse MS data.

Hydroxyl Radical Protein Footprinting: A Mass Spectrometry-Based Structural Method for Studying the Higher Order Structure of Proteins.

Hydroxyl radical protein footprinting (HRPF) coupled to mass spectrometry has been successfully used to investigate a plethora of protein-related questions. The method, which utilizes hydroxyl radicals to oxidatively modify solvent-accessible amino acids, can inform on protein interaction sites and regions of conformational change. Hydroxyl radical-based footprinting was originally developed to study nucleic acids, but coupling the method with mass spectrometry has enabled the study of proteins. The method has undergone several advancements since its inception that have increased its utility for more varied applications such as protein folding and the study of biotherapeutics. In addition, recent innovations have led to the study of increasingly complex systems including cell lysates and intact cells. Technological advances have also increased throughput and allowed for better control of experimental conditions. In this review, we provide a brief history of the field of HRPF and detail recent innovations and applications in the field.

The utilization of the search engine, Bolt, to decrease search time and increase peptide identifications in hydroxyl radical protein footprinting-based workflows.

Hydroxyl radical protein footprinting (HRPF) utilizes hydroxyl radicals to covalently modify solvent exposed regions of proteins. When coupled with mass spectrometry, HRPF can provide insightful information on protein structural changes including inside cells. However, the complex mixture of proteins and modifications makes identification a complicated task. To search all of the HRPF-induced modification combinations across the full proteome, requires substantial computational power and still can take days to search. To drastically decrease processing time and improve identifications, a novel cloud-based search engine, Bolt, was used to search for HRPF modifications in comparison to a commonly used search engine, Sequest. A 35% increase in the identification of modified peptides was observed in Bolt compared to Sequest with a decrease in computation time.

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins.

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein structure, protein-protein interactions, or protein-ligand interactions. HRPF utilizes hydroxyl radicals (▪OH) to irreversibly label a protein's solvent accessible surface. The inherent complexity, cost, and hazardous nature of performing HRPF have substantially limited broad-based adoption in biopharma. These factors include: 1) the use of complicated, dangerous, and expensive lasers that demand substantial safety precautions; and 2) the irreproducibility of HRPF caused by background scavenging of ▪OH that limit comparative studies. This publication provides a protocol for operation of a laser-free HRPF system. This laser-free HRPF system utilizes a high energy, high-pressure plasma light source flash oxidation technology with in-line radical dosimetry. The plasma light source is safer, easier to use, and more efficient in generating hydroxyl radicals than laser-based HRPF systems, and the in-line radical dosimeter increases the reproducibility of studies. Combined, the laser-free HRPF system addresses and surmounts the mentioned shortcomings and limitations of laser-based techniques.

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

This protocol presents a method to use inline radical dosimetry and a plasma light source to perform flash oxidation protein footprinting. This method replaces the hazardous UV laser to simplify and improve the reproducibility of fast photochemical oxidation of protein studies.

Cryo-EM reveals the architecture of placental malaria VAR2CSA and provides molecular insight into chondroitin sulfate binding

Placental malaria can have severe consequences for both mother and child and effective vaccines are lacking. Parasite-infected red blood cells sequester in the placenta through interaction between parasite-expressed protein VAR2CSA and the glycosaminoglycan chondroitin sulfate A (CS) abundantly present in the intervillous space. Here, we report cryo-EM structures of the VAR2CSA ectodomain at up to 3.1 Å resolution revealing an overall V-shaped architecture and a complex domain organization. Notably, the surface displays a single significantly electropositive patch, compatible with binding of negatively charged CS. Using molecular docking and molecular dynamics simulations as well as comparative hydroxyl radical protein foot-printing of VAR2CSA in complex with placental CS, we identify the CS-binding groove, intersecting with the positively charged patch of the central VAR2CSA structure. We identify distinctive conserved structural features upholding the macro-molecular domain complex and CS binding capacity of VAR2CSA as well as divergent elements possibly allowing immune escape at or near the CS binding site. These observations will support rational design of second-generation placental malaria vaccines.

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

Hydroxyl radical protein footprinting (HRPF) is a powerful and flexible technique for probing changes in protein topography. With the development of the fast photochemical oxidation of proteins (FPOP), it became possible for researchers to perform HRPF in their laboratory on a very short time scale. While FPOP has grown significantly in popularity since its inception, adoption remains limited due to technical and safety issues involved in the operation of a hazardous Class IV UV laser and irreproducibility often caused by improper laser operation and/or differential radical scavenging by various sample components. Here, we present a new integrated FOX (Flash OXidation) Protein Footprinting System. This platform delivers sample via flow injection to a facile and safe-to-use high-pressure flash lamp with a flash duration of 10 μs fwhm. Integrated optics collect the radiant light and focus it into the lumen of a capillary flow cell. An inline radical dosimeter measures the hydroxyl radical dose delivered and allows for real-time compensation for differential radical scavenging. A programmable fraction collector collects and quenches only the sample that received the desired effective hydroxyl radical dose, diverting the carrier liquid and improperly oxidized sample to waste. We demonstrate the utility of the FOX Protein Footprinting System by determining the epitope of TNFα recognized by adalimumab. We successfully identify the surface of the protein that serves as the epitope for adalimumab, identifying four of the five regions previously noted by X-ray crystallography while seeing no changes in peptides not involved in the epitope interface. The FOX Protein Footprinting System allows for FPOP-like experiments with real-time dosimetry in a safe, compact, and integrated benchtop platform.

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

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.

Mapping the Epitope Interactions of Trastuzumab Via Plasma Induced Modification of Biomolecules (PLIMB)

Epitope mapping is the process of identifying the site(s) of an antigen where an antibody binds. Knowledge of epitopes facilitates drug design and patent applications; hence it is a key element of vaccine and drug development. Hydroxyl radical protein footprinting coupled with mass spectrometry offers opportunities to map antibody-binding regions of antigens with greater simplicity and increased throughput over x-ray crystallography. We report here the use of hydroxyl radical protein footprinting via Plasma Induced Modification of Biomolecules (PLIMB) to characterize the epitopes of trastuzumab (Herceptin) bound to receptor tyrosine-protein kinase erbB-2 (HER2). The protein samples were exposed to plasma for hydroxyl radical labeling, the labeled sites were identified using mass spectrometry, and the data was analyzed using Byos (Protein Metrics). The PLIMB data is compared to previously published results that determined the epitope of trastuzumab using x-ray crystallography.