Confirmation of disulfide linkages in adalimumab using electron activated dissociation (EAD) 


Featuring the SCIEX ZenoTOF 7600 system with EAD and Protein Metrics Inc. software

Zoe Zhang1 , Jason Causon2 , Pavel Ryumin2 , Takashi Baba2 , Bill Loyd2 , Kerstin Pohl1
1SCIEX, US; 2SCIEX, Canada

Abstract


The data presented in this technical note show the confirmation of disulfide-linked peptides alongside the confirmation of the sequence for a biotherapeutic monoclonal antibody (mAb). The analysis of disulfide-linked peptides was performed using rapid data dependent acquisition (DDA) and automated data interpretation with Protein Metrics Inc. software as part of a disulfide mapping analysis. With this workflow, regular and advanced characterization leveraging the novel electron activated dissociation (EAD)-based fragmentation1, 2 is achievable in one injection, enabling a streamlined characterization accessible to every user level.

Ensuring drug safety and efficacy is essential for biotherapeutics and drives the need for in-depth characterization during their development. This includes the identification and the localization of post-translational modifications (PTMs), especially the characterization of disulfide linkages. Since disulfide bonds are critical for maintaining the three-dimensional structure and function of biomolecules3 , elucidating the connections between cysteine residues is essential for validating disulfide design and understanding the higher order structure of biotherapeutics. Different analytical and biophysical methods have been employed for the analysis of disulfide linkages, such as Edman degradation.4 With the wide adoption of mass spectrometry, bottom-up approaches with tandem mass spectrometry have become the method of choice.5 However, disulfide bonds prevent effective fragmentation around the linked cysteine residues in collision induced dissociation (CID), which limits the information available for identifying the disulfide-bound peptides as well as disulfide patterns. Although alternative fragmentation mechanisms exist, these techniques can suffer from long reaction times, low sensitivity and lack of reproducibility.

Here, a new fragmentation type based on EAD1, 2 is demonstrated for the confirmation of disulfide-linked peptides from a commercial mAb. The data were acquired using an untargeted 10 Hz DDA method and interpreted with Protein Metrics Inc. software. This workflow demonstrates a routine approach for the streamlined characterization of disulfide-linked peptides with a new level of data quality.
 

Key features of EAD with the SCIEX ZenoTOF 7600 system
 

  • New depths of peptide mapping analysis: EAD with fast DDA enables alternative fragmentation for routine, in-depth analysis of next generation protein therapeutics and standard mAbs 

  • Higher levels of structural information: Changing the mechanism of fragmentation by tuning the electron energy may provide a higher level of structural information, particularly for disulfide linked peptides and sequence confirmation
     
  • Higher MS/MS sensitivity: Increased detection of fragments (5 to 10 fold) using the Zeno trap enables higher confidence in data assignment 

  • High reproducibility: Reproducible fragmentation with EAD for singly, doubly, and multiply charged ions enables analysis of more precursors than other alternative and low reproducibility fragmentation techniques 

  • Streamlined and easy-to-use: Fully automated data acquisition in DDA mode using EAD with SCIEX OS software, and automated data interpretation with Byos software (Protein Metrics Inc.) simplifies the entire user experience 

Figure 1. The SCIEX ZenoTOF 7600 system.