biostudies-literatureSchulte DEuropean Research CouncilDutch Research Council (NWO)RP101322https://www.ebi.ac.uk/biostudies/studies/S-EPMC12017766biostudies-literatureUnknown14Antibodies are a major component of adaptive immunity against invading pathogens. Here, we explore possibilities for an analytical approach to characterize the antigen-specific antibody repertoire directly from the secreted proteins in convalescent serum. This approach aims to perform simultaneous antibody sequencing and epitope mapping using a combination of single particle cryo-electron microscopy (cryoEM) 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 cryoEM reconseLifeSimultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry.PMC1201776610.13039/100019180101077640Gravitation 2013 BOO,Institute for Chemical Immunology (ICI; 024.002.009)Kall LSiborova MSnijder JSchulte DfalseSimultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry.Antibodies are a major component of adaptive immunity against invading pathogens. Here, we explore possibilities for an analytical approach to characterize the antigen-specific antibody repertoire directly from the secreted proteins in convalescent serum. This approach aims to perform simultaneous antibody sequencing and epitope mapping using a combination of single particle cryo-electron microscopy (cryoEM) 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 cryoEM recons2025-01-01T00:00:00Z2025 Apr2026-07-15T11:01:32.449Z2025-07-10T03:08:58.142ZS-EPMC120177664026625210.7554/eLife.101322