WILD®

WILD® stands for w-ion isoleucine (Ile/I) and leucine (Leu, L) determination. I and L are isobaric amino acids, meaning they have identical masses, which makes it difficult to differentiate them using traditional mass spectrometry. To elucidate the mass of proteins, digested peptides are run through a mass spectrometer where they undergo collisions that induce fragmentation of the peptide backbone, generating N- and C-terminal ions [1] (Figure 1). C-terminal z-ions contain the sidechains of the peptide backbone. When fragmentation of the z-ion sidechains occurs, w-ions are generated. Though I and L are isobaric, their w-ions are not.

Figure 1. Although Leucine (Leu, L) and Isoleucine (Ile, I) share the same mass (113.08406 Da), our WILD® technology employs controlled fragmentation in a mass spectrometer to cleave off different parts of their side chains. This controlled fragmentation yields different w-ions, which are used to distinguish I and L.

The w-ion was first reported in 1987 by Johnson, Martin and Biemann [1]. However, it was mostly known in academic circles. Rapid Novor became the first to commercially offer a w-ion-based I/L determination in antibody sequencing. At Rapid Novor, we employ proprietary protein chemistry and state-of-the-art Orbitrap Fusion instruments that can perform electron-transfer high energy collision dissociation (EThcD). EThcD of z-ions yields w-ions [2]. Our protein chemistry allows us to consistently and successfully determine all I/Ls in our customers’ samples. With WILD®, we uncover I and L residues accurately and with high throughput.

Having been the first ones to introduce WILD®  for antibody sequencing commercially, we have successfully completed the most I/L determination projects with unparalleled accuracy and throughput.

Figure 2. A spectrum of a peptide from an antibody’s variable region with 3 I/L in its sequence. Our proprietary protein chemistry and EThcD allow us to generate w- and z-ions; our accurate and automated software then calculates the difference between these ions to annotate the sequence.

At the end of a long research day, why guess when you can have certainty?

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References

[1]R. S. Johnson, S. A. Martin and K. Biemann, “Novel Fragmentation Process of Peptides by Collision-Induced Decomposition in a Tandem Mass Spectrometer: Differentiation of Leucine and Isoleucine,” Analytical Chemistry, vol. 59, no. 21, pp. 2621-2625, 1987.
[2]S. S. Zhokhov, S. V. Kovalyov, T. Y. Samgina and A. T. Lebedev, “An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides,” Journal of the American Society for Mass Spectrometry, vol. 28, no. 8, pp. 1600-1611, 2017.