Application Note

Differentiation Of Leucine And Isoleucine Using Electron Activated Dissociation (EAD)

Source: SCIEX

By Zoe Zhang, Pavel Ryumin, Takashi Baba, Jason Causon, Bill Lloyd, and Kerstin Pohl, SCIEX

Amino Acid GettyImages-1289685060

Ensuring drug safety and efficacy is essential for biotherapeutic development, which drives the need for in-depth characterization. Confirmation of the protein sequence is a standard requirement for all protein therapeutics by regulatory agencies. Although mass spectrometry (MS) has been mostly adapted for sequence verification, differentiating Leu/Ile remains a challenge. These two isomeric amino acids have the same molecular weight and an MS/MS spectrum obtained from collision-induced dissociation (CID) cannot tell them apart. Thus, Edman degradation is still widely employed today, which is reagent expensive and time consuming.

Alternative fragmentation techniques have been reported to introduce secondary fragmentation, resulting in different losses of the side chain from Leu and Ile.The derived signature ions can be used to discriminate between these two amino acids. However, previous approaches required extensive pre-characterization in order to perform MS3 experiments. The use of nano-liquid chromatography (LC), offline fractionation or infusion was also common to enhance sensitivity, but they lacked reproducibility and throughput. With more and more protein therapeutics in the market and in development, the need to distinguish Leu and Ile in an easy manner has dramatically increased. In addition, it is likely that more analytical questions will need to be answered which require technologies better able to elucidate complex structural moieties.

The data presented in this work show the streamlined identification of Leu and Ile as part of a general peptide mapping study. Data were acquired in a fast, automatic and sensitive manner using data-dependent acquisition (DDA) with Zeno EAD, with streamlined data interpretation utilizing Protein Metrics Inc. software.

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