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1.
J Mass Spectrom ; 59(7): e5064, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38873895

RESUMO

Mass spectrometry-based approaches to assess protein conformation have become widely utilized due to their sensitivity, low sample requirements, and broad applicability to proteins regardless of size and environment. Their wide applicability and sensitivity also make these techniques suitable for the analysis of complex mixtures of proteins, and thus, they have been applied at the cell and even the simple organism levels. These works are impressive, but they predominately employ "bottom-up" workflows and require proteolytic digestion prior to analysis. Once digested, it is not possible to distinguish the proteoform from which any single peptide is derived and therefore, one cannot associate distal-in primary structure-concurrent post-translational modifications (PTMs) or covalent labels, as they would be found on separate peptides. Thus, analyses via bottom-up proteomics report the average PTM status and higher-order structure (HOS) of all existing proteoforms. Second, these works predominately employ promiscuous reagents to probe protein HOS. While this does lead to improved conformational resolution, the formation of many products can divide the signal associated with low-copy number proteins below signal-to-noise thresholds and complicate the bioinformatic analysis of these already challenging systems. In this perspective, I further detail these limitations and discuss the positives and negatives of top-down proteomics as an alternative.


Assuntos
Espectrometria de Massas , Conformação Proteica , Pegadas de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas , Proteômica , Espectrometria de Massas/métodos , Pegadas de Proteínas/métodos , Proteínas/química , Proteínas/análise , Proteômica/métodos
2.
Anal Chem ; 96(19): 7386-7393, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38698660

RESUMO

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.


Assuntos
Mapeamento de Epitopos , Receptor ErbB-2 , Trastuzumab , Humanos , Mapeamento de Epitopos/métodos , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , Trastuzumab/química , Alquilação , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Halogenação , Pegadas de Proteínas/métodos , Complexo Antígeno-Anticorpo/química
3.
Anal Chem ; 96(23): 9693-9703, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38815160

RESUMO

Specific amino acid footprinting mass spectrometry (MS) is an increasingly utilized method for elucidating protein higher order structure (HOS). It does this by adding to certain amino acid residues a mass tag, whose reaction extent depends on solvent accessibility and microenvironment of the protein. Unlike reactive free radicals and carbenes, these specific footprinters react slower than protein unfolding. Thus, their footprinting, under certain conditions, provokes structural changes to the protein, leading to labeling on non-native structures. It is critical to establish conditions (i.e., reagent concentrations, time of reaction) to ensure that the structure of the protein following footprinting remains native. Here, we compare the efficacy of five methods in assessing protein HOS following footprinting at the intact protein level and then further localize the perturbation at the peptide level. Three are MS-based methods that provide dose-response plot analysis, evaluation of Poisson distributions of precursor and products, and determination of the average number of modifications. These MS-based methods reliably and effectively indicate HOS perturbation at the intact protein level, whereas spectroscopic methods (circular dichroism (CD) and dynamic light scattering (DLS)) are less sensitive in monitoring subtle HOS perturbation caused by footprinting. Evaluation of HOS at the peptide level indicates regions that are sensitive to localized perturbations. Peptide-level analysis also provides higher resolution of the HOS perturbation, and we recommend using it for future footprinting studies. Overall, this work shows conclusive evidence for HOS perturbation caused by footprinting. Implementation of quality control workflows can identify conditions to avoid the perturbation, for footprinting, allowing accurate and reliable identification of protein structural changes that accompany, for example, ligand interactions, mutations, and changes in solution environment.


Assuntos
Proteínas , Proteínas/química , Espectrometria de Massas , Pegadas de Proteínas/métodos , Conformação Proteica , Aminoácidos/química , Dicroísmo Circular
4.
Anal Chem ; 96(19): 7566-7576, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38684118

RESUMO

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRASG12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRASG12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.


Assuntos
Metano , Metano/análogos & derivados , Metano/química , Humanos , Pegadas de Proteínas/métodos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ligação Proteica , Espectrometria de Massas
5.
Biochim Biophys Acta Proteins Proteom ; 1872(4): 141011, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38499233

RESUMO

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 hydrogen­deuterium 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.


Assuntos
Mapeamento de Epitopos , Radical Hidroxila , Pegadas de Proteínas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Mapeamento de Epitopos/métodos , Pegadas de Proteínas/métodos , SARS-CoV-2/imunologia , SARS-CoV-2/química , Radical Hidroxila/química , Humanos , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Ligação Proteica , COVID-19/virologia , COVID-19/imunologia , Sítios de Ligação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/química , Espectrometria de Massas/métodos , Domínios Proteicos
6.
J Am Soc Mass Spectrom ; 35(3): 476-486, 2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38335063

RESUMO

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.


Assuntos
Radical Hidroxila , Síncrotrons , Humanos , Raios X , Radical Hidroxila/química , Pegadas de Proteínas/métodos , Espectrometria de Massas em Tandem , Oxirredução
7.
Anal Chem ; 95(50): 18316-18325, 2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-38049117

RESUMO

Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a quantitative method of hydroxyl radical generation via Fe(II)-ethylenediaminetetraacetic acid (EDTA)-catalyzed Fenton chemistry that provides ready access to protein oxidative footprinting using equipment commonly found in research and process control laboratories. Robust and reproducible dose-dependent oxidation of protein samples is observed and quantitated by mass spectrometry with as fine a single residue resolution. An oxidation analysis of lysozyme provides a readily accessible benchmark for our method. The efficacy of our oxidation method is demonstrated by mapping the interface of a RAS-monobody complex, the surface of the NIST mAb, and the interface between PRC2 complex components. These studies are executed using standard laboratory tools and a few pennies of reagents; the mass spectrometry analysis can be streamlined to map the protein structure with single amino acid residue resolution.


Assuntos
Radical Hidroxila , Proteínas , Ácido Edético/química , Radical Hidroxila/química , Proteínas/análise , Pegadas de Proteínas/métodos , Estresse Oxidativo , Oxirredução
8.
J Am Soc Mass Spectrom ; 34(12): 2864-2867, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-37971787

RESUMO

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.


Assuntos
Radical Hidroxila , Pegadas de Proteínas , Radical Hidroxila/química , Catalase , Pegadas de Proteínas/métodos , Proteínas/química , Oxirredução
9.
J Am Soc Mass Spectrom ; 34(12): 2700-2710, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-37967285

RESUMO

Membrane proteins (MPs) play a crucial role in cell signaling, molecular transport, and catalysis and thus are at the heart of designing pharmacological targets. Although structural characterization of MPs at the molecular level is essential to elucidate their biological function, it poses a significant challenge for structural biology. Although mass spectrometry-based protein footprinting may be developed into a powerful approach for studying MPs, the hydrophobic character of membrane regions makes structural characterization difficult using water-soluble footprinting reagents. Herein, we evaluated a small series of MS-based photoactivated iodine reagents with different hydrophobicities. We used tip sonication to facilitate diffusion into micelles, thus enhancing reagent access to the hydrophobic core of MPs. Quantification of the modification extent in hydrophilic extracellular and hydrophobic transmembrane domains provides structurally sensitive information at the residue-level as measured by proteolysis and LC-MS/MS for a model MP, vitamin K epoxide reductase (VKOR). It also reveals a relationship between the reagent hydrophobicity and its preferential labeling sites in the local environment. The outcome should guide the future development of chemical probes for MPs and promote a direction for relatively high-throughput information-rich characterization of MPs in biochemistry and drug discovery.


Assuntos
Pegadas de Proteínas , Espectrometria de Massas em Tandem , Indicadores e Reagentes , Cromatografia Líquida , Proteínas de Membrana/química , Interações Hidrofóbicas e Hidrofílicas
10.
Eur J Mass Spectrom (Chichester) ; 29(5-6): 292-302, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37750197

RESUMO

Irreversible protein footprinting is a mass spectrometry-based approach in which solvent-accessible sites of a protein are modified to assess high-order protein structure. Structural insights can be gained by determining the position and extents of modification. The usual approach to obtain the "footprint" is to analyze the protein through bottom-up LC-MS/MS. In this approach, the proteins are digested to yield a mixture of peptides that are then separated by LC before locating the modification sites by MS/MS. This process consumes substantial amounts of time and is difficult to accelerate for applications that require quick and high-throughput analysis. Here, we describe employing matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) to analyze a footprinted small test protein (ubiquitin) via a top-down approach. Matrix-assisted laser desorption/ionization is easily adapted for high-throughput analysis, and top-down strategies can avoid lengthy proteolysis and LC separation. We optimized the method with model peptides and then demonstrated its feasibility on ubiquitin submitted to two types of footprinting. We found that MALDI ISD can produce a comprehensive set of fragment ions for small proteins, affording footprinting information in a fast manner and giving results that agree with the established methods, and serve as a rough measure of protein solvent accessibility. To assist in the implementation of the MALDI approach, we developed a method of processing top-down ISD data.


Assuntos
Pegadas de Proteínas , Espectrometria de Massas em Tandem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cromatografia Líquida , Proteínas/química , Peptídeos/química , Ubiquitina/química , Solventes
11.
Biochem Biophys Res Commun ; 671: 343-349, 2023 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-37329657

RESUMO

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.


Assuntos
Radical Hidroxila , Pegadas de Proteínas , Pegadas de Proteínas/métodos , Conformação Proteica , Proteínas/química , Espectrometria de Massas/métodos
12.
Anal Chem ; 95(26): 10119-10126, 2023 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-37351860

RESUMO

Protein footprinting mass spectrometry probes protein higher order structure and dynamics by labeling amino acid side-chains or backbone amides as a function of solvent accessibility. One category of footprinting uses residue-specific, irreversible covalent modifications, affording flexibility of sample processing for bottom-up analysis. Although several specific amino acid footprinting technologies are becoming established in structural proteomics, there remains a need to assess fundamental properties of new reagents before their application. Often, footprinting reagents are applied to complex or novel protein systems soon after their discovery and sometimes without a thorough investigation of potential downsides of the reagent. In this work, we assemble and test a validation workflow that utilizes cyclic peptides and a model protein to characterize benzoyl fluoride, a recently published, next-generation nucleophile footprinter. The workflow includes the characterization of potential side-chain reactive groups, reaction "quench" efficacies, reagent considerations and caveats (e.g., buffer pH), residue-specific kinetics compared to those of established reagents, and protein-wide characterization of modification sites with considerations for proteolysis. The proposed workflow serves as a starting point for improved footprinting reagent discovery, validation, and introduction, the aspects of which we recommend before applying to unknown protein systems.


Assuntos
Aminoácidos , Proteínas , Aminoácidos/química , Fluxo de Trabalho , Proteínas/química , Espectrometria de Massas/métodos , Pegadas de Proteínas/métodos
13.
J Am Soc Mass Spectrom ; 34(3): 417-425, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36700916

RESUMO

Many cancer drugs fail at treating solid epithelial tumors with hypoxia and insufficient drug penetration thought to be contributing factors to the observed chemoresistance. Owing to this, it is imperative to evaluate potential cancer drugs in conditions as close to in vivo as possible, which is not always done. To address this, we developed a mass spectrometry-based protein footprinting method for exploring the impact of hypoxia on protein in 3D colorectal cancer cells. Our group has previously extended the protein footprinting method fast photochemical oxidation of proteins (FPOP) for live cell analysis (IC-FPOP); however, this is the first application of IC-FPOP in a 3D cancer model. In this study, we perform IC-FPOP on intact spheroids (Spheroid-FPOP) using a modified version of the static platform incubator with an XY movable stage (PIXY) FPOP platform. We detected modification in each of three spheroid layers, even the hypoxic core. Pathway analysis revealed protein modifications in over 10 distinct protein pathways, including some involved in protein ubiquitination; a process modulated in cancer pathologies. These results demonstrate the feasibility of Spheroid-FPOP to be utilized as a tool to interrogate protein interactions within a native tumor microenvironment.


Assuntos
Antineoplásicos , Neoplasias , Humanos , Pegadas de Proteínas/métodos , Proteínas/análise , Espectrometria de Massas/métodos , Oxirredução , Esferoides Celulares/química , Microambiente Tumoral
14.
Anal Chem ; 94(28): 9993-10002, 2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35797180

RESUMO

Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in a mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. Here, we investigate the possibility to analyze a specifically selected population of only singly oxidized proteins. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is the selection of a suitable method of ion isolation, excitation, and fragmentation. Here, we employ and compare collision-induced dissociation, electron-transfer dissociation, and electron-capture dissociation combined with multi-continuous accumulation of selected ions. A singly oxidized subpopulation of FPOP-labeled ubiquitin was used to optimize the method. The top-down approach in FPOP is limited to smaller proteins, but its usefulness was demonstrated by using it to visualize structural changes induced by co-factor removal from the holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in good agreement, which indicates that monitoring a singly oxidized FPOP ion population by the top-down approach is a functional workflow for oxidative protein footprinting.


Assuntos
Elétrons , Pegadas de Proteínas , Mioglobina/química , Estresse Oxidativo , Conformação Proteica , Pegadas de Proteínas/métodos
16.
Anal Chem ; 94(27): 9819-9825, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35763792

RESUMO

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.


Assuntos
Aminoácidos , Pegadas de Proteínas , Aminoácidos/química , Radical Hidroxila/química , Oxirredução , Conformação Proteica , Pegadas de Proteínas/métodos , Proteínas/análise , Água
17.
Commun Biol ; 5(1): 452, 2022 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-35551273

RESUMO

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.


Assuntos
Pegadas de Proteínas , Proteínas , Simulação por Computador , Radical Hidroxila/química , Domínios de Imunoglobulina , Espectrometria de Massas , Pegadas de Proteínas/métodos , Proteínas/química
18.
Anal Chem ; 94(3): 1520-1524, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35019278

RESUMO

Protein footprinting mass spectrometry (MS), an emerging approach to elucidate higher-order structure (HOS) and binding, benefits from the iterative development of reaction strategies to expand the covalent labeling toolbox. Herein, we introduce a footprinting reagent for nucleophiles and demonstrate its efficacy for differential covalent labeling MS analysis. Benzoyl fluoride (BF), although reactive with water, is more practical for modifying nucleophilic functional groups than other acid halides and serves as an acyl-transfer reagent for proteins. BF is 10 times more reactive with phenolic Tyr than the current generation nucleophile footprinter. BF modifies, in addition to Tyr, Lys, His, and the N-terminus, weak nucleophiles Ser and Thr, for which few footprinters exist, imparting broad applicability with a range of nucleophiles. We applied benzoylation to a model Ser- and Thr-rich protein-ligand binding system without perturbing the protein HOS. This efficacious footprinting method expands the toolbox of reagents and provides promise for future reaction strategies including possibly membrane proteins.


Assuntos
Pegadas de Proteínas , Proteômica , Indicadores e Reagentes , Espectrometria de Massas/métodos , Proteínas de Membrana , Pegadas de Proteínas/métodos , Proteômica/métodos
19.
Chem Rev ; 122(8): 7532-7561, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-34633178

RESUMO

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.


Assuntos
Radical Hidroxila , Pegadas de Proteínas , Radical Hidroxila/química , Espectrometria de Massas , Dobramento de Proteína , Pegadas de Proteínas/métodos , Proteínas/química
20.
Biotechnol Bioeng ; 119(1): 211-225, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34687215

RESUMO

In this article, a systematic workflow was formulated and implemented to understand selectivity differences and preferred binding patches for bispecific monoclonal antibodies (mAbs) and their parental mAbs on three multimodal cation exchange resin systems. This workflow incorporates chromatographic screening of the parent mAbs and their fragments at various pH followed by surface property mapping and protein footprinting using covalent labeling followed by liquid chromatography-mass spectrometry analysis. The chromatography screens on multimodal resins with the intact mAbs indicated enhanced selectivity as compared to single-mode interaction systems. While the bispecific antibody (bsAb) eluted between the two parental mAbs on most of the resins, the retention of the bispecific transitioned from co-eluting with one parental mAb to the other parental mAb on Capto MMC. To investigate the contribution of different domains, mAb fragments were evaluated and the results indicated that the interactions were likely dominated by the Fab domain at higher pH. Protein surface property maps were then employed to hypothesize the potential preferred binding patches in the solvent-exposed regions of the parental Fabs. Finally, protein footprinting was carried out with the parental mAbs and the bsAb in the bound and unbound states at pH 7.5 to identify the preferred binding patches. Results with the intact mAb analysis supported the hypothesis that interactions with the resins were primarily driven by the residues in the Fab fragments and not the Fc. Furthermore, peptide mapping data indicated that the light chain may be playing a more important role in the higher binding of Parent A as compared with Parent B in these resin systems. Finally, results with the bsAb indicated that both halves of the molecule contributed to binding with the resins, albeit with subtle differences as compared to the parental mAbs. The workflow presented in this paper lays the foundation to systematically study the chromatographic selectivity of large multidomain molecules which can provide insights into improved biomanufacturability and expedited downstream bioprocess development.


Assuntos
Anticorpos Biespecíficos , Cromatografia Líquida/métodos , Pegadas de Proteínas/métodos , Anticorpos Biespecíficos/análise , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Biespecíficos/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Ligação Proteica , Propriedades de Superfície
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