Hydrogen deuterium exchange mass spectrometry hdxms is an ideal method to probe a proteins higherorder structure. Coales sj, tuske sj, tomasso jc, hamuro y 2009 epitope mapping by amide hydrogendeuterium exchange coupled with immobilization of antibody, online proteolysis, liquid. Identification and characterization of antibody binding. Hydrogen deuterium exchange mass spectrometry hdxms is a powerful biophysical technique being increasingly applied to a wide variety of problems. Hydrogen deuterium exchange hdxms when diluted in heavy water d2o, backbone hydrogens of amino acids exchange with deuterium at varying kinetics rates depending upon their hydrogen bonding and solvent accessibility. Hydrogendeuterium exchange epitope mapping reveals distinct neutralizing mechanisms for two monoclonal antibodies against. Mapping proteinligand interactions with proteolyticdigestion, hydrogendeuterium exchangemass spectrometry.
Here we highlight the mass spectrometry ms based approach of. Understanding hydrogen deuterium exchange hdx lcms. For epitope mapping, hd exchange is performed by exposing the peptide or. Epitope mapping of diverse influenza hemagglutinin drug.
Hydrogendeuterium exchange mass spectrometry hdxms is an ideal method to probe a proteins higherorder structure. Techniques that can rapidly map binding epitopes are of particular interest because. Hydrogendeuterium exchange mass spectrometry hdxms is a powerful approach for mapping protein folding, proteinligand interactions, proteinprotein interactions, and conformational changes. Hdx technology for protein conformation studies youtube. Highresolution epitope mapping by hx ms reveals the pathogenic.
Hydrogen deuterium exchange hdx ms works in many applications, including understanding how smallmolecule therapeutics bind to protein targets, and performing epitope mapping. Integrated software platform for analyzing hydrogendeuterium exchange and oxidative footprinting data. The results obtained with hydrogendeuterium exchange mass spectrometry hdx ms were compared with the previous findings made using the excisionextraction ms approach. We used a hydrogendeuterium exchange mass spectrometry hx ms method to. Contact us to learn more about our proprietary covalent labeling technology and how we. Hydrogen deuterium exchange hdx mass spectrometry ms is a powerful tool for studying the dynamics of higher order structure of proteinbased therapeutics. Epitope mapping and higher order structure slideshare uses cookies to improve functionality and. Creative biolabs has used hydrogendeuterium hd exchange on a research scale for many years and with the advancement of sample handling and data analysis software, now we are dedicated to. Epitope mapping of an interleukin receptor for three therapeutic. Hydrogen deuterium exchange mass spectrometry hdx ms.
Hydrogendeuterium exchange mass spectrometry hdxms is an increasingly popular protein characterization technology. Hydrogen deuterium exchange hdx lcms analysis is a workflow that is increasingly used to increase the knowledge of biotherapeutics and biosimilars, find out how this analysis is. Hydrogendeuterium exchange mass spectrometry hdxms has emerged as an alternative method for epitopeparatope mapping with important applications in drug discovery. An introduction to bcell epitope mapping and in silico.
At various time points the deuteriumhydrogen exchange is quenched at 0c. Pdf epitope mapping by epitope excision, hydrogendeuterium. Epitope mapping is the process of experimentally identifying the binding site, or epitope, of an antibody on its target antigen usually, on a protein. Red arrows highlight three software commonly used for sequence analysis. We performed epitope mapping for an antigen with molecular weight of 200 kda and we achieved more than 90% sequence coverage. Deuterium exchange was determined with hdexaminer software. Hydrogendeuterium exchange mass spectrometry laboratory. Cremap epitope mapping service by hydrogendeuterium.
Epitope mapping of a monoclonal antibody directed against. With the recent availability of commercial automated systems, hydrogen deuterium exchange hdx is. Cremap epitope mapping service by hydrogendeuterium exchange. Quarrybio provides high throughput, high resolution, and low cost epitope mapping and protein structure analyses.
Application of amide hydrogendeuterium exchange mass. Hydrogen deuterium exchange hdxms when diluted in heavy water d2o, backbone hydrogens of amino acids exchange with deuterium at varying kinetics rates depending upon their. Review an overview of hydrogen deuterium exchange mass spectrometry hdxms in drug discovery glenn r. Among all the analytical techniques available for epitope mapping studies, hydrogendeuterium exchange mass spectrometry hdxms is. Hydrogendeuterium exchange epitope mapping reveals distinct. The mascot software matrix science was used to search msms data in. Covalx epitope mapping techniques hydrogen deuterium exchange hdxms hydrogen deuterium exchange hdxms measures the accessibility of hydrogen molecules in the backbone of a protein. Covalx epitope mapping techniques hydrogen deuterium exchange hdxms hydrogen deuterium exchange hdxms measures the accessibility of hydrogen molecules in the backbone of a protein structure. Here, we use hydrogendeuterium exchange mass spectrometry.
It relies on the accurate measurement of the degree of labeling of the protein by deuterated hydrogen. Hydrogendeuterium exchange also called hd or hd exchange is a chemical reaction in which a covalently bonded hydrogen atom is replaced by a deuterium atom, or vice versa. Epitope mapping by amide hydrogendeuterium exchange. Hydrogendeuterium exchange mass spectrometry hdx ms has become an important technique for the analysis of protein structure and dynamics.
Hydrogen deuterium exchange mass spectrometry hdxms takes advantage of this phenomena by exchanging the protons from the protein with deuterium in deuterated solutions. Hydrogen deuterium exchange mass spectrometry protein footprinting is a major research theme of the wu research resource in mass spectrometry. An antigen is mixed a deuterium buffer in solution, and the mixture was incubated for a series of predetermined time points. Hydrogendeuterium exchange mass spectrometry for probing. When the antigen is complexed with the antibody, this deuterium uptake rate is altered in the epitope regions. Covalx epitope mapping service by hdxms allows confomrational and linear.
The onexchanged antigen is loaded onto an antibody column. System licor and quantified by densitometry using imagej software. Epitope mapping by epitope excision, hydrogendeuterium exchange, and peptidepanning techniques combined with in silico analysis. Highresolution hx ms mapping of an antigenic epitope. Hdxms automation robotics and software to rapidly characterize the binding. During analysis, both the unbound antigen and the bound antibodyantigen complex are incubated in deuterated water in order to exchange any amide hydrogens from exposed amino acids of the proteins backbone. For peptide deuterium uptake, the first two residues and prolines were excluded from calculations. With the advent of fully automated sample preparation robots for hydrogendeuterium exchange coupled to mass spectrometry hdxms, this method has become.
The graphs show the deuterium uptake data over 30 min labeling for factor h binding protein fhbp fragments in the absence red or presence blue of monoclonal antibody mab 12c1. Example of epitope mapping by hydrogen deuterium exchange mass spectrometry hdxms. We have experience with many different antigens or proteins, some of them with high molecular weight and multiple glycosylation. Show full abstract the mass spectrometry ms based approach of hydrogendeuterium exchange hdx and describe its utility in epitope mapping. Key words b cell epitope, epitope excision, hydrogendeuterium exchange. Hydrogen deuterium exchange hdx is an experimental technique to obtain structural data on proteins. Epitope mapping of an interleukin receptor for three therapeutic antibodies by hydrogendeuterium exchange mass spectrometry 8 eric largy, caroline cajot, and arnaud delobel among all the analytical. Robust method for full structural characterization. Astra software, including the protein conjugate analysis function, was used for data analysis. Epitope mapping by epitope excision, hydrogendeuterium.
Epitope mapping by amide hydrogendeuterium exchange coupled with immobilization of antibody, on. It is widely applicable and is capable of monitoring the dynamic properties of a. The acquity uplc mclass system with hdx technology leverages uplc separations and highresolution ms to answer important questions about changes in protein conformation using. The rate of hydrogentodeuterium exchange within the amide hydrogen on the backbone of biotherapeutics provides solvent accessibility. The higher order structure of protein therapeutics can be interrogated with hydrogendeuterium exchange mass spectrometry hdxms. The use of hydrogendeuterium exchange mass spectrometry in epitope mapping article in chimica oggi 311. In recent years, various techniques have been described for epitope mapping, each with its own strengths and weaknesses. Epitope mapping of an antibody receptor using hydrogen. Suppressing allostery in epitope mapping experiments using. The results obtained with hydrogendeuterium exchange mass spectrometry.
Hydrogen deuterium exchange mass spectrometry hdxms is a powerful technique to study protein dynamics, protein folding, proteinprotein interactions, protein small molecule interactions, as well as. Recommendations for performing, interpreting and reporting. The results showed that for all three software packages. Protein characterization by hydrogendeuterium exchange mass spectrometry. Hydrogendeuterium exchange mass spectrometry for probing higher order structure of protein therapeutics. To achieve this result, different epitopemapping approaches have been widely. This ligand offers a new vector for attacking solid tumor cancers ref. The method can analyze proteins conformationaldynamic properties in. Used hdxms and mutagenesis to find the epitope of proliferating cell nuclear antigen pcna and a previously unknown ligand, tip. Mapping proteinligand interactions with proteolytic. Contact us to learn more about our proprietary covalent labeling technology and how we can help you with your protein challenges. Hydrogen deuterium exchange with mass spec and hdx ms. Hydrogendeuterium exchange mass spectrometry hdxms has become a popular method for analysis of the conformational dynamics and interactions of pro. Creative biolabs has massive experience on epitope mapping programs over.
Subsequently, epitope mapping by hydrogendeuterium exchange mass spectrometry hdxms was performed. This fast, costeffective hdxms epitope mapping approach. It is widely applicable and is capable of monitoring the dynamic properties of a protein at the submolecular level in solution. Data analysis remains a bottleneck in the workflow. With this technology, deuterium in a solution replaces the amide hydrogen in a biomolecules backbone, and ms measures these changes. Integrated software platform for analyzing hydrogen. The use of hydrogendeuterium exchange mass spectrometry.
457 187 1350 617 1031 566 1158 807 874 50 86 209 1087 1550 719 1359 330 548 1054 356 1563 1099 1053 500 973 1104 819 701 1281 304 54 1514 319 285 455 417 1044 899 992 1174 203 90 1015 373 744