Fig. 1. Schematic overview of IgGs structures and the here used sequencing workflow. (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). 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). Besides different numbers of HC-bridging disulfides, IgG 1–4 subclasses also differ by the disulfide bond between the light chain (LC) and the HC: directly above the hinge for IgG1, mediated by a more N-terminal HC cysteine for IgG 2–4. Additionally, the IgG3 hinge region is longer owing to an insertion that is repeated three times, as indicated with a black line above the sequence. (B) The purified F(ab′)2 portions of all IgG subclasses are subjected to ECD-MS, which preferentially fragments the LC and HC between their disulfide-bridged loops. Clearly separated low m/z c-ions and complementary high m/z bridged z-ions are formed, enabling the straightforward interpretation of fragment ion ladders beneficial for de novo IgG sequencing.
