The first step in the workflow involves the separation by proteolytic cleavage of the IgG CDR-containing F(ab′) 2 from the glycosylated constant Fc using the IdeS enzyme, which cleaves (scissors) just below the disulfides bridges (blue) between the heavy chains (HCs). (A) Distinctive structures of IgG1 and IgG2-4, all consisting of two variable regions (colored) with three antigen-binding Complementarity Determining Regions (CDRs) each, and one constant region (black). 1 Schematic overview of IgGs structures and the here used sequencing workflow. Additionally, IgG subclasses exhibit distinctive disulfide bridging with a different number of disulfide bonds in the hinge region and different positioning of disulfides between LC and HC. Only specific stretches of the HC regions differ between the subclasses: mainly the hinge region and the N-terminal side of the HC region below the hinge (Fc). The HC constant domain, on the other hand, is divided into four subclasses, namely IgG1, IgG2, IgG3, and IgG4, having between 83% and 96% sequence similarity. The LC constant domain can be of two types, namely kappa (κ) or lambda (λ). Structurally, IgGs are made of four disulfide-bridged polypeptide chains: two identical light chains (LC) of 25 kDa and two identical heavy chains (HC) of approximately 50 kDa ( Fig. Shortening the route from the discovery of an antibody to the production of a functional equivalent is therefore of utmost importance, certainly in present times. by the SARS-CoV-2, Dengue 6 or Ebola virus, 7 can lead to a substantial reduction in the mortality rate of newly infected patients. 5 Therapeutic antibodies reflecting those of an individual having survived a severe infection, e.g. 1–4 The high selectivity of immunoglobulins G (IgGs) for a single antigen has made this class of antibodies of great value in basic research, but even more important in molecular medicine. Introduction Antibodies are key molecules of our immune system and therefore of great interest to many researchers, both in academia and industry. 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. 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. Pure ECD, without additional collisional activation, leads to straightforward-to-read sequence tags covering the CDR3 of both the light and heavy chains. 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. 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. 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. Therefore, methods focusing solely on the variable regions and providing unambiguous sequence reads are strongly advantageous. These constant parts of IgG do not yield any useful information in attempts to sequence antibodies de novo. Although incredibly diverse in specificity, millions of unique Immunoglobulin G (IgG) molecules in the human antibody repertoire share most of their amino acid sequence.
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