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Selectivity over coverage in de novo sequencing of IgGs

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Abstract

Although incredibly diverse in specificity, millions of unique Immunoglobulin G (IgG) molecules in the human antibody repertoire share most of their amino acid sequence. These constant parts of IgG do not yield any useful information in attempts to sequence antibodies de novo. Therefore, methods focusing solely on the variable regions and providing unambiguous sequence reads are strongly advantageous. We report a mass spectrometry-based method that uses electron capture dissociation (ECD) to provide straightforward-to-read sequence ladders for the variable parts of both the light and heavy chains, with a preference for the functionally important CDR3. We optimized this method on the therapeutic antibody Trastuzumab and demonstrate its applicability on two monoclonal quartets of the four IgG subclasses, IgG1, IgG2, IgG3 and IgG4. The method is based on proteolytically separating the variable F(ab′)2 part from the conserved Fc part, whereafter the F(ab′)2 portions are mass-analyzed and fragmented by ECD. Pure ECD, without additional collisional activation, leads to straightforward-to-read sequence tags covering the CDR3 of both the light and heavy chains. Using molecular modelling and structural analysis, we discuss and explain this selective fragmentation behavior and describe how structural features of the different IgG subclasses lead to distinct fragmentation patterns. Overall, we foresee that pure ECD on F(ab′)2 or Fab molecules can become a valuable tool for the de novo sequencing of serum antibodies.

Graphical abstract: Selectivity over coverage in de novo sequencing of IgGs

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Supplementary files

Article information


Submitted
19 Jun 2020
Accepted
06 Oct 2020
First published
06 Oct 2020

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2020, Advance Article
Article type
Edge Article

Selectivity over coverage in de novo sequencing of IgGs

M. A. den Boer, J. Greisch, S. Tamara, A. Bondt and A. J. R. Heck, Chem. Sci., 2020, Advance Article , DOI: 10.1039/D0SC03438J

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