MSACL 2023 Abstract

Differentiation of Leucine and Isoleucine Using Electron Activated Dissociation (EAD)

Casey Burrows (1), Zoe Zhang (1) , Pavel Ryumin (2) , Takashi Baba (2) , Jason Causon (2) , Bill Lloyd (2) , Kerstin Pohl (1)
(1) Sciex US, (2) Sciex Canada

Casey Burrows (Presenter) SCIEX

Presenter Bio: Casey Burrows received a bachelor’s degree in chemistry from Georgia Southern University in 2011. From 2013-2015, he worked as an analytical chemist for the Florida Department of Agriculture and Consumer Services to develop, modify, validate, and migrate LC/MS analytical methods for determining contaminates in food matrices. Casey is now based in Georgia where he works as a member of our clinical and forensic toxicology groups as a field application specialist since 2015.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction:
Here, an unambiguous differentiation of leucine (Leu) and isoleucine (Ile) in peptides derived from a monoclonal antibody (mAb) therapeutic was achieved utilizing a new fragmentation type based on electron activated dissociation (EAD). This workflow demonstrates the combination of routine characterization and elucidation of challenging residues in one single analysis, without the need for sample specific optimization.

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 precharacterization 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.

Methods:
The adalimumab sample was denaturated with 7.2 M guanidine hydrochloride, 100 mM Tris buffer pH 7.2, followed by reduction with 10 mM DL-dithiothreitol and alkylation with 30 mM iodoacetamide. Digestion was performed with trypsin/Lys-C enzyme at 37 °C for 16h. 3 µl (4 µg) of the trypsin/Lys-C digest was separated with a CSH C18 column (2.1×100 mm, 1.7 μm, 130 Å, Waters) using an ExionLC system. The mobile phase A consisted of water with 0.1% formic acid, while the organic phase B was acetonitrile 0.1% formic acid. A gradient profile was used at a flow rate of 350 μL/min. The column temperature was maintained at 50°C. Data were acquired with an information dependent acquisition (IDA) method using the SCIEX ZenoTOF 7600 system. The electron energy for the alternative fragmentation in the EAD cell was set to a value of 7 eV. Data were processed using Byos software (Protein Metrics Inc.).

Results:
A peptide mapping analysis of a mAb was performed capable of answering in-depth characterization questions like Leu/Ile differentiation, while maintaining sensitivity and ease-ofuse. An analytical flow LC setup and a DDA approach with Zeno EAD on the SCIEX ZenoTOF 7600 system were used. Robustness and throughput are achieved by using analytical flow rates, while the DDA method set-up further enhance ease-of-use by avoiding compound-specific method optimization. In addition, the Zeno trap enhances the detection of fragments enabling a confident assignment.6 This breakthrough technology realizes the desire of an easy, and at the same time comprehensive, method for advanced characterization in the biopharmaceutical industry.

Conclusions:
Unambiguous differentiation between Ile and Leu in one single DDA run was achieved by using Zeno EAD on the SCIEX ZenoTOF 7600 system with automatic data interpretation by Protein Metrics Inc. software. This workflow proposes a streamlined solution for distinguishing isomers previously thought a challenge by LC-MS/MS for years.