SmartEnzymes in de novo Sequencing of Antibodies
Two complementary determining regions (CDR3) are considered major determinants of antigen-binding specificity that give rise to the human immunoglobulin repertoire with billions of unique antibodies. For de novo sequencing of the human repertoire, circulating antibodies can be analyzed by mass spectrometry after proteolytic cleavage. Complex mixtures such as plasma derived samples are however challenging to analyze due to the increased complexity that may prevent accurate assignments.
In this paper, scientists at Utrecht University explored de novo sequencing by analysis at the antibody subunit level using electron capture dissociation (ECD) and mass spectrometry. To decrease complexity and target the variable CDR3s regions, antibodies were digested to obtain homogenous F(ab’)2 or Fab subunits using the FabRICATOR® or FabALACTICA® enzymes respectively. The unique capability of FabRICATOR to digest human IgG1-4 allowed the scietists to process multiple antibodies for this analytical approach.
Sample complexity was decreased by removing the Fc part containing PTMs including glycosylation and lysine clipping, while the F(ab’)2 fragments were subjected to fragmentation and mass spectrometry analysis. The use of EDC allowed for amino acid sequence ladders spanning the CDR3 region of both the light and heavy chains, with gaps only observed for proline containing sequences. By studying different antibodies and subclasses, the authors were able to observe correlations between subclass-specific features and fragmentation patterns.
This work by den Boer et al. is a first step in predicting the fragmentation patterns observed in ECD fragmentation of IgGs, which may greatly contribute to future tools for de novo sequencing of antibodies.
Generate F(ab’)2 and Fc/2 from IgG.
Generate Fab and Fc from human IgG1.