Leu/Ile Differentiation Service

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Precisely determine Leu/Ile to reduce your expression cost!

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Leu/Ile Differentiation Service Key Features:

  • Design and perform LC-MS/MS analysis (≥50μg of a purified protein)
  • Guarantee accuracy by multiple proofs
  • Guarantee two-week turn-around and service quality

Why is Leu/Ile differentiation needed?

Although leucine (Leu) and isoleucine (Ile) share exactly the same molecular weight, their different structures may dramatically influence protein functions. To thoroughly characterize a protein of interest, especially a therapeutic protein, researchers have to cautiously examine each Leu/Ile position in the protein sequence. Particularly, for Leu/Ile residues in and near CDRs of an antibody, flipping between Leu/Ile may result in significant differences in its biochemical properties.

Our Leu/Ile differentiation service provides a precise identification of Leu and Ile residues during protein sequencing and to help dramatically reduce the cost in the subsequent expression stage. For instance, a target protein which contains 4 undetermined Leu/Ile residues would need up to 16 constructs expressed to fully test which residue is correct. This does not even take codon combinations into account! With our Leu/Ile differentiation service, you no longer need to guess and repeatedly test, but be 100% certain from the start.

For the purpose of protein sequence validation, our Leu/Ile differentiation service can help you confirm the expression of Leu/Ile in your protein of interest and discover potential sequence variants.

How to differentiate Leu/Ile?

Our Leu/Ile differentiation service consists of customized LC-MS/MS experiments using the EThcD fragmentation method and subsequent data analysis. Based on state-of-the-art MS instruments, advanced method settings, and enhanced in-house software tools, satisfaction is guaranteed to meet our customers’ needs. The precise Leu/Ile information is retrieved from:

1. Leu and Ile residues are generally considered to be indistinguishable by traditional MS. However, w-ions of isoleucine and leucine generated by certain MS methods can distinguish them 1,2,3;

(Image cited from Ref. 2)


2. (Optional) The use of enzymes is important as a diagnostic and research tool because of the specificity they exhibit. By exploiting enzymatic cleavage rules, we can use enzymes such as chymotrypsin or pepsin, for example, to differentiate between Leu/Ile residues;

3. (Optional) Statistical comparison of homologous databases for conservative sites in the mAb constant regions and framework regions.


1. Johnson, R. S., Martin, S., Biemann, K., Stults, J. T., & Watson, J. T. Novel fragmentation process of peptides by collision-induced decomposition in a tandem mass spectrometer: differentiation of leucine and isoleucine. Anal Chem. 1987 Nov 1;59(21):2621-5.
2. Xiao, Y., Vecchi, M. M., & Wen, D. Distinguishing between Leucine and Isoleucine by Integrated LC-MS Analysis using an Orbitrap Fusion Mass Spectrometer. Anal Chem. 2016 Nov 1;88(21):10757-10766.
3. Zhokhov, S. S., Kovalyov, S. V., Samgina, T. Y., & Lebedev, A. T. An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides. J Am Soc Mass Spectrom. 2017 Aug;28(8):1600-1611.