Isoleucine (Ile, I) and leucine (Leu, L) are isobaric residues or amino acids with the same mass (Figure 1). Conventional mass spectrometry-based proteomics cannot be easily used to distinguish between Ile and Leu. 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.

Figure 1. Leucine (Leu, L), and isoleucine (Ile, I) amino acids’ chemical structure, molecular weight (Da) (mass which includes the extra H2O) and monoisotopic residue mass (Da) (does not include the hydroxyl group of the carboxylic acid nor one of the hydrogens on the amine).

Why is it important to distinguish between Ile and Leu?

Each antibody has 6 complementarity determining regions (CDRs), 3 on each heavy and light chain, perhaps the most important sites for antibody/antigen interaction (Figure 2) [1]. Incorrect identification of a single amino acid in the CDRs can lead to expressing an antibody with different binding and biological activity. This is particularly the case for CDR-H3, which is the most variable and unique of all CDRs. 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.

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 Ile and Leu residues are very rare. Ninety-seven percent of antibodies will contain at least one I/L residue in their CDRs, and more than half of the antibodies we sequenced using our popular REmAb® sequencing service contained at least 4 Ile or Leu residues in their CDRs [3]. Moreover, approximately 10% of sequenced antibodies contained more than 7 Ile or Leu

Figure 2. 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.

Take a moment to imagine

If you had an antibody with a CDR containing 9 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®

Figure 3. 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®).

What about other isobaric amino acids?

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 [3]. We also digest our samples with multiple proteases to yield many overlapping peptides that can help us verify the entire protein sequence with great accuracy, and redundancy [4]. Why guess when you can have certainty? Send us your antibody samples and keep working with confidence. You can learn more about how WILD®works here.

Maria Rosales Gerpe, Ph.D
Scientific Writer
Rapid Novor, Inc.


  1. Sela-Culang, I., et al., The Structural Basis of Antibody-Antigen Recognition. Frontiers in Immunology, 2013. 4.
  2. McDonald, Z.L., Q; Stajduhar, A; Taylor, P; Krieger, JR; Ma, B, Large Scale Study of the W-ion Isoleucine and Leucine Determination (WILD™) Method in Antibody De Novo Protein Sequencing, in 66th American Society for Mass Spectrometry. 2018: San Diego.
  3. Ma, B., Novor: real-time peptide de novo sequencing software. Journal of the American Society for Mass Spectrometry 2015. 26(11): p. 1885-1894.
  4. Le Bihan, T., et al. Increased De Novo Protein Sequencing Coverage with Optimal Protease Cocktail. in American Association for Mass Spectrometry. 2019. Atlanta.