Development of MS based Routine Epitope Mapping Techniques

Daniel Nyberg Larsen

doi:10.21996/6rjh-0636

Development of MS based Routine Epitope Mapping Techniques

Bidragets oversatte titel: Udvikling af MS baseret rutine epitope mapping teknikker

Daniel Nyberg Larsen

Publikation: Afhandling › Ph.d.-afhandling

Abstract

Proteininteraktion er en del af alle funktioner i kroppen, lige fra immunsystemet til cellens grundlæggende funktioner. Interaktionen kan studeres vha. en række forskellige teknikker, f.eks. spektroskopimetoder, ”linear epitope mapping” og massespektrometri baserede metoder. Her udnyttede vi en massespektrometrisk metode. Den anvendte epitope mapping metode er baseret på overflademærkningskonceptet, hvor hydroxylradikaler er ansvarlige for at modificere overfladen. Det analyserede protein behandles derefter alene eller sammen med et bindende protein, f.eks. et antistof. Ud fra sammenligningen mellem de to betingelser kan et bindingssted bestemmes.

Adskillige metoder, der anvender hydroxylradikaler til overflademærkningsteknikker, er tilgængelige. De er dog alle relativt dyre at anskaffe og har ikke været kommercielt tilgængelige gennem en kontraktforskningsorganisation (CRO). Vi satte os derfor for at lave en metode baseret på hydroxyl radikal protein footprinting (HRPF) ved hjælp af Fenton kemi. Målet var at udvikle en hurtig, reproducerbar og implementerbar metode, som krævede mindst mulig tid at udføre.

For at nå disse kriterier blev arbejdet fokuseret på tidligere kendte teknikker og ideer fra andre forskningsfelter samt automatisering. Derfor blev en dataproces udviklet for at opnå relativt hurtig datahåndtering og analyse. Endvidere blev der indbygget en rapportfunktion til hurtig dataanalyse, som samtidig kunne leveres til fremtidige kunder. Automatiseringen blev ikke kun udført i datahåndteringen, men også på prøveforberedelsen. Den komplette protokol blev testet på Opentrons OT2 pipetteringsrobot, for at viser et proof-of-concept. Til artiklen præsenteret i afhandlingen blev der anvendt en semi-automatiseret metode, hvor oxidationsprotokollen blev udført i OT2.

Metoden blev testet på to biologiske systemer, det murine CD163 og det receptorbindingsdomæne af SARS-CoV-2 Spike protein (RBD). Epitope mapping metode for murint CD163 viste et lovende proof-of-concept experiment, og et bindingssted blev bestemt. I artiklen blev to varianter af RBD undersøgt og viste et fælles bindingssted. Bindingsstedet for RBD Alpha og RBD Delta imod anti-RBD-antistoffer var i overensstemmelse med den kendte litteratur for neutraliserende antistoffer. Ved at introducere TMT-mærkning og ikke-bindende negative kontrolantistoffer blev 40% af de signifikant ændrede peptider udelukket, hvilket resulterede i stærkere og mere pålidelige data.

Metoden endte med at spare ~80% arbejdstimer på prøvehåndteringen og tilsvarende på datahåndteringen. Implementeringsomkostningerne for vores metode er ~80% lavere sammenlignet med andre applikationer, der bruger HRPF, f.eks. PLIMB. Samtidig har vores applikation den ekstra mulighed for fuldstændig automatisering af metoden. Yderligere optimering af protokollen kunne være fordelagtig, men proof-of-concept-metoden viste lovende resultater. Det arbejde, der præsenteres her, beviste derfor, at epitope mapping ved Fenton-kemi kunne være en brugbar metode for CRO eller antistofproducenter.

Bidragets oversatte titel	Udvikling af MS baseret rutine epitope mapping teknikker
Originalsprog	Engelsk
Bevilgende institution	Syddansk Universitet
Vejledere/rådgivere	Højrup, Peter, Vejleder Graversen, Jonas Heilskov, Vejleder Kaczmarek, Jakub Zbigniew, Vejleder
Eksterne samarbejdspartnere	Ovodan Biotech A/S
Dato for forsvar	30. jan. 2024
Udgiver	Syddansk Universitet. Det Naturvidenskabelige Fakultet
DOI	https://doi.org/10.21996/6rjh-0636
Status	Udgivet - 9. jan. 2024

Note vedr. afhandling

Afhandlingen kan læses på SDUs bibliotek. 

Emneord

Proteinkemi
Epitope mapping
Massespektrometri

Dokumenter og links

10.21996/6rjh-0636

1 Tidsskriftartikel

Epitope mapping of SARS-CoV-2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls
Larsen, D. N., Kaczmarek, J. Z., Palarasah, Y., Graversen, J. H. & Højrup, P., 1. jul. 2024, I: BBA Proteins and Proteomics. 1872, 4, 141011.
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

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abstract = "Protein interaction is part of all functions in the body, from the immune system to the basic functions of the cell. Proteins can also be targeted by bacteria or viruses prior to infection. The interaction can be studied by a range of techniques e.g., spectroscopy methods, linear epitope mapping, and mass spectrometric based methods. Here we exploited a mass spectrometric based method. The epitope mapping method utilized is based on the surface labeling concept, where hydroxyl radicals are responsible for modifying the surface. The analyzed protein is then treated alone or together with a binding protein e.g., an antibody. From the comparison between the two conditions a binding site can be determined. Several methods using hydroxyl radicals for surface labeling techniques are available. However, they are all relatively expensive to acquire and have not been commercially available through a contract research organization. We therefore set out to make a method based on hydroxyl radical protein footprinting (HRPF) using Fenton chemistry. The aim was to develop a fast, reproducible, and implementable method, which required the least amount of time to run. To achieve these criteria, the work was focused on previously known techniques and ideas from other research fields and automatization. Consequently, a data process was developed to achieve relatively fast data handling and analysis. Furthermore, a report function was incorporated for fast data analysis and could be delivered to future customers. The automatization was not only performed in the data handling but also on the sample preparation. The complete protocol was tested on the Opentrons OT2 pipetting robot, showing a proof-of-concept. For the article presented in the thesis, a semi-automatized method was utilized, with the oxidation protocol performed in the OT2.The method was tested on two biological systems, the murine CD163 and the receptor binding domain of SARS-CoV-2 Spike protein (RBD). The epitope mapping method for murine CD163 showed promising proof-of-concept and a binding site was determined. Within the article, two variants of RBD were investigated and showed a common binding site. The binding site of RBD Alpha and RBD Delta to anti-RBD antibodies were in agreement with the known literature for neutralizing antibodies. Furthermore, by introducing TMT labeling and non-binding negative control antibodies, 40% of the significantly changes peptides were excluded, resulting in stronger and more reliable data.The method ended up saving ~80% working hours on the sample handling and similarly on the data handling. The implementation cost of our method is ~80% lower compared to other applications using HRPF e.g., PLIMB, with the additional possibility for complete automatization of the method. Further optimization of the protocol could be beneficial, however, the proof-of-concept method showed promising results. The work presented here, therefore, proved that epitope mapping by Fenton chemistry could be a viable method for contract research organization or antibody producers.",

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AB - Protein interaction is part of all functions in the body, from the immune system to the basic functions of the cell. Proteins can also be targeted by bacteria or viruses prior to infection. The interaction can be studied by a range of techniques e.g., spectroscopy methods, linear epitope mapping, and mass spectrometric based methods. Here we exploited a mass spectrometric based method. The epitope mapping method utilized is based on the surface labeling concept, where hydroxyl radicals are responsible for modifying the surface. The analyzed protein is then treated alone or together with a binding protein e.g., an antibody. From the comparison between the two conditions a binding site can be determined. Several methods using hydroxyl radicals for surface labeling techniques are available. However, they are all relatively expensive to acquire and have not been commercially available through a contract research organization. We therefore set out to make a method based on hydroxyl radical protein footprinting (HRPF) using Fenton chemistry. The aim was to develop a fast, reproducible, and implementable method, which required the least amount of time to run. To achieve these criteria, the work was focused on previously known techniques and ideas from other research fields and automatization. Consequently, a data process was developed to achieve relatively fast data handling and analysis. Furthermore, a report function was incorporated for fast data analysis and could be delivered to future customers. The automatization was not only performed in the data handling but also on the sample preparation. The complete protocol was tested on the Opentrons OT2 pipetting robot, showing a proof-of-concept. For the article presented in the thesis, a semi-automatized method was utilized, with the oxidation protocol performed in the OT2.The method was tested on two biological systems, the murine CD163 and the receptor binding domain of SARS-CoV-2 Spike protein (RBD). The epitope mapping method for murine CD163 showed promising proof-of-concept and a binding site was determined. Within the article, two variants of RBD were investigated and showed a common binding site. The binding site of RBD Alpha and RBD Delta to anti-RBD antibodies were in agreement with the known literature for neutralizing antibodies. Furthermore, by introducing TMT labeling and non-binding negative control antibodies, 40% of the significantly changes peptides were excluded, resulting in stronger and more reliable data.The method ended up saving ~80% working hours on the sample handling and similarly on the data handling. The implementation cost of our method is ~80% lower compared to other applications using HRPF e.g., PLIMB, with the additional possibility for complete automatization of the method. Further optimization of the protocol could be beneficial, however, the proof-of-concept method showed promising results. The work presented here, therefore, proved that epitope mapping by Fenton chemistry could be a viable method for contract research organization or antibody producers.

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KW - Massespektrometri

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DO - 10.21996/6rjh-0636

M3 - Ph.D. thesis

PB - Syddansk Universitet. Det Naturvidenskabelige Fakultet

ER -

Development of MS based Routine Epitope Mapping Techniques

Abstract

Note vedr. afhandling

Emneord

Dokumenter og links

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

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