Do-It-Yourself De Novo Antibody Sequencing Workflow that Achieves Complete Accuracy of the Variable Regions

peer-reviewed · Journal of Proteome Research · 2025

peer-reviewed · Journal of Proteome Research · 2025. Meng-Ting He et al. Antibodies are widely used as research tools or therapeutic agents. Knowing the sequences of the variable…
Date 2025-06-06
Type peer-reviewed
Venue Journal of Proteome Research
Publisher American Chemical Society (ACS)
Contribution algorithm
DOI 10.1021/acs.jproteome.5c00210
Citations (OpenAlex) 2
Venue 2-year citedness 3.83

Abstract

Antibodies are widely used as research tools or therapeutic agents. Knowing the sequences of the variable regions of an antibody─both the heavy chain and the light chain─is a prerequisite for the production of recombinant antibodies. Mass spectrometry-based de novo sequencing is a frequently used, and sometimes the only approach to gaining this information. Here, we describe a workflow that enables accurate sequence determination of monoclonal antibodies based on mass spectrometry data and freely available software tools. This workflow, which we developed using a homemade anti-FLAG monoclonal antibody as a reference sample, achieved 100% accuracy of the variable regions with clear distinction between leucine (L) and isoleucine (I). Using this workflow, we successfully decoded a monoclonal anti-HA antibody, for which we had no prior knowledge of its sequence. Based on the de novo sequencing result, we generated a recombinant anti-HA antibody, and demonstrated that it has the same specificity, sensitivity, and affinity as the commercial antibody.

Authors

  1. Meng-Ting He · Beijing Normal University, National Institute of Biological Sciences, Beijing, Tsinghua University
  2. Ning Li (Beijing) · National Institute of Biological Sciences, Beijing, Tsinghua University
  3. Jian-Hua Wang · Changping Laboratory
  4. Zhi-Zhong Wei · National Institute of Biological Sciences, Beijing, Tsinghua University
  5. Jie Feng · National Institute of Biological Sciences, Beijing, Tsinghua University
  6. Wenting Li · Bioinformatics Solutions (Canada), Bioinformatics Solutions Inc., University of Waterloo
  7. Jian-Hua Sui · National Institute of Biological Sciences, Beijing, Tsinghua University
  8. Niu Huang · National Institute of Biological Sciences, Beijing, Tsinghua University
  9. Meng-Qiu Dong · National Institute of Biological Sciences, Beijing, Tsinghua University

Methods and tools

  • DIY de novo antibody sequencing workflow: Assembled from off-the-shelf parts rather than new software: multi-protease digestion, complementary fragmentation and existing de novo tools, combined into a protocol that reached complete accuracy across antibody variable regions and decoded an unknown anti-HA antibody.

Cites (30)

Cited by (3)

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