A new generation of nanobody research tools using improved mass spectrometry-based discovery methods

Peter C Fridy; Ryan J Farrell; Kelly R Molloy; Sarah Keegan; Junjie Wang; Erica Y Jacobs; Yinyin Li; Jill Trivedi; Viren Sehgal; David Fenyö; Zhuhao Wu; Brian T Chait; Michael P Rout

doi:10.1016/j.jbc.2024.107623

A new generation of nanobody research tools using improved mass spectrometry-based discovery methods

J Biol Chem. 2024 Sep;300(9):107623. doi: 10.1016/j.jbc.2024.107623. Epub 2024 Aug 2.

Authors

Peter C Fridy¹, Ryan J Farrell², Kelly R Molloy³, Sarah Keegan⁴, Junjie Wang³, Erica Y Jacobs⁵, Yinyin Li³, Jill Trivedi¹, Viren Sehgal¹, David Fenyö⁴, Zhuhao Wu⁶, Brian T Chait⁷, Michael P Rout⁸

Affiliations

¹ Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York, USA.
² Laboratory of Brain Development and Repair, The Rockefeller University, New York, New York, USA; Department of Biochemistry, Weill Cornell Medicine, New York, New York, USA.
³ Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York, USA.
⁴ Department of Biochemistry and Molecular Pharmacology, Institute for Systems Genetics, NYU Grossman School of Medicine, New York, New York, USA.
⁵ Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York, USA; Chemistry Department, St John's University, Queens, New York, USA.
⁶ Laboratory of Brain Development and Repair, The Rockefeller University, New York, New York, USA.
⁷ Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York, USA. Electronic address: chait@rockefeller.edu.
⁸ Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York, USA. Electronic address: rout@rockefeller.edu.

Abstract

Single-domain antibodies ("nanobodies") derived from the variable region of camelid heavy-chain only antibody variants have proven to be widely useful tools for research, therapeutic, and diagnostic applications. In addition to traditional display techniques, methods to generate nanobodies using direct detection by mass spectrometry and DNA sequencing have been highly effective. However, certain technical challenges have limited widespread application. We have optimized a new pipeline for this approach that greatly improves screening sensitivity, depth of antibody coverage, antigen compatibility, and overall hit rate and affinity. We have applied this improved methodology to generate significantly higher affinity nanobody repertoires against widely used targets in biological research-i.e., GFP, tdTomato, GST, and mouse, rabbit, and goat immunoglobulin G. We have characterized these reagents in affinity isolations and tissue immunofluorescence microscopy, identifying those that are optimal for these particularly demanding applications, and engineering dimeric constructs for ultra-high affinity. This study thus provides new nanobody tools directly applicable to a wide variety of research problems, and improved techniques enabling future nanobody development against diverse targets.

Keywords: antibody engineering; immunohistochemistry; mass spectrometry; monoclonal antibody; single-domain antibody.

MeSH terms

Animals
Goats
Humans
Immunoglobulin G / chemistry
Immunoglobulin G / immunology
Mass Spectrometry* / methods
Mice
Rabbits
Single-Domain Antibodies* / chemistry
Single-Domain Antibodies* / immunology

Substances

Single-Domain Antibodies
Immunoglobulin G