Simultaneous polyclonal antibody sequencing and epitope mapping by cryo-electron microscopy and mass spectrometry

Antibodies are a major component of adaptive immunity against invading pathogens. In this presentation, we will describe an analytical approach to characterize the antigen-specific antibody repertoire directly from the secreted proteins in convalescent serum. This approach enables simultaneous antibody sequencing and epitope mapping using a combination of single particle cryo-electron microscopy (cryo-EM) and bottom-up proteomics techniques based on mass spectrometry (LC-MS/MS). We evaluate the performance of the deep-learning tool ModelAngelo in determining de novo antibody sequences directly from reconstructed 3D volumes of antibody-antigen complexes. 

We demonstrate that while map quality is a critical bottleneck, it is possible to sequence antibody variable domains from cryo-EM reconstructions with accuracies of up to 80-90%. While the rate of errors exceeds the typical levels of somatic hypermutation, we show that the ModelAngelo-derived sequences can be used to assign the used V-genes. This provides a functional guide to assemble de novo peptides from LC-MS/MS data more accurately and improves the tolerance to a background of polyclonal antibody sequences.

We show that such EM-derived templates indeed improve MS-based sequencing accuracy in the context of complex antibody mixtures and that publicly available EMPEM reconstructions are of sufficient quality to leverage this approach. This proof-of-principle offers a promising perspective to integrate cryo-EM and MS methods for a comprehensive characterization of the antibody repertoire on both sequence and epitope levels.

Learning Objectives

• Understand the significance of antibodies in adaptive immunity against pathogens.
• Learn about the combined use of cryo-electron microscopy and mass spectrometry for antibody sequencing and epitope mapping.
• Learn about a deep-learning tool in sequencing antibody variable domains from cryo-EM reconstructions.
   

Presenter: Joost Snijder, PhD (Assistant Professor, Utrecht University)

Joost Snijder uses structural proteomics to study virus-host interactions, with a focus on the antiviral antibody response. He obtained his PhD with Albert Heck in 2015 developing native mass spectrometry to study virus structure and assembly. With the support of EMBO and an NWO Rubicon grant, he then joined the lab of David Veesler at the University of Washington in Seattle. Here he learned single particle cryo electron microscopy to study virus structure and map the epitopes of antiviral antibodies. In addition, he used MS-based glycoproteomics to map the glycan shield of viral antigens. In 2019 he returned to Utrecht with the support of the NWO Gravitation program Institute for Chemical Immunology to study virus replication and develop new methods to sequence antibodies directly by mass spec. In 2023, he was awarded an ERC Starting Grant to study the polyclonal antibody response against flaviviruses with structural proteomics techniques