Because they share the same mass, isoleucine and leucine are known as isobaric amino acids. Conventional mass spectrometry-based proteomics cannot be easily used to distinguish between isoleucine and leucine. However, in 2016, Rapid Novor became the first to commercially introduce a service, w-ion isoleucine-leucine determination (WILD® ), to distinguish between Ile and Leu in de novo antibody sequencing.
Why is it important to distinguish between Isoleucine and Leucine in Antibodies?
Each antibody has six complementarity-determining regions (CDRs): three on each heavy and light chain, perhaps the most important sites for antibody/antigen interaction. Incorrect identification of a single amino acid in the CDRs can lead to expressing an antibody with unexpected binding and biological activity. This is particularly the case for CDR-H3, which is the most variable and unique of all CDRs (1). For this reason, it is crucial to be able to identify the sequence of the CDRs with 100% certainty, including differentiating between Ile and Leu residues. The latter is especially important because Ile and Leu are frequently found in these hypervariable regions (2).
Figure 3. Representative diagram of protein structure of an IgG class antibody. From N-terminus to C-terminus, the antigen-binding region (Fab) contains a variable (orange) and constant (blue) region for both heavy (VH and CH1-3) and light chains (VL and CL), respectively. The variable regions (VH and VL) (inset box) contain the complementarity determining regions (CDRs1-3) interspersed through framework regions (FR1-3). The Fab region is separated from the crystallizable fragment (Fc) domain by a disulphide bridge or hinge (red) with IdeS digestion. The Fc is composed of constant domains from the two heavy chains.
We have found that isoleucine and leucine are frequently observed in monoclonal antibodies. In 2017, we sequenced 200 monoclonal antibodies, and found that CDRs with no isoleucine or leucine residues are very rare (3). Ninety-seven percent of antibodies will contain at least one isoleucine or leucine residue in their CDRs, and more than half of the antibodies we sequenced using our popular REmAb® sequencing service contained at least four isoleucine or leucine residues in their CDRs. Moreover, approximately 10% of sequenced antibodies contained more than seven isoleucine or leucine residues.
Figure 4. Frequency of antibodies expressing isoleucine or leucine in their complementarity determining regions (CDRs). A total of 200 monoclonal antibodies were sequenced using our de novo protein sequencing platform (REmAb®).
Take a moment to imagine. If an antibody had a CDR containing nine isoleucine/leucine positions, you would be required to express 512 forms just to confirm the full antibody sequence. Why resort to guesswork when you can have certainty with WILD®?
Other isobaric amino acids like isoleucine and leucine
In addition to WILD®, REmAb® can also identify other isobaric residues. By taking into account proteolysis and peptide fragmentation patterns, our machine learning software assigns confidence scores to each amino acid and peptide derived from mass spectral data. We also digest our samples with multiple proteases to yield many overlapping peptides (4) that can help us verify the entire protein sequence with great accuracy, and redundancy.