A new electron activated dissociation (EAD) approach for comprehensive glycopeptide analysis of therapeutic proteins

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

Fang Wang1 , Jenny Albernese1 , Zoe Zhang1 , Pavel Ryumin2 , Takashi Baba2 , Jason Causon2 , Kerstin Pohl1
SCIEX, US; 2SCIEX, Canada 


The data presented here demonstrate the advantage of the novel electron activated dissociation (EAD)1,2 over traditionally used collision induced dissociation (CID) with regards to glycopeptide characterization and localization in a peptide mapping workflow. In addition, streamlined, advanced characterization in one injection is being offered through high speed, highly reproducible, alternative fragmentation.3-6 This solution takes peptide mapping experiments to a new level.

As biotherapeutics are becoming more complex, challenges for in-depth characterization increase simultaneously. Their characterization involves a myriad of analytical methods which include, but are not limited to, amino acid sequence confirmation and identification and localization of post-translational modifications (PTMs).3,6 Glycosylations in particular are PTMs frequently considered critical quality attributes, as their composition and levels can affect the effector functions and the in vivo half-life of a biotherapeutic product. Glycosylations tend to be present in a highly heterogenous manner in terms of structure and abundancies, increasing the complexity of their analysis.7-8 A robust glycopeptide mapping solution can assign the glycosylation sites, determine the compositions of the attached glycans and estimate their relative abundances.

EAD, a newly developed dissociation approach which is unique to the SCIEX ZenoTOF 7600 system, allows for a tunable electron energy that produces varied fragmentation patterns for a wide range of peptides in a peptide mapping workflow.1,2 The resulting MS/MS fragment ions from glycopeptides showed peptide backbone fragment ions with glycosylation remaining intact. This allows for accurate localization of the linked glycans along with confident identification of the peptide through high MS/MS sequence coverage of the peptide backbone. Zeno EAD (Figure 1) enables fast and sensitive data dependent acquisition (DDA). This approach overcomes challenges of alternative fragmentation such as long reaction times and low sensitivity. Trastuzumab was used as an example to show how users of all levels can apply this technology for their streamlined characterization of glycopeptides, improving efficiency and understanding of their biotherapeutics.

Key features of 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 well-suited for glycopeptide characterization
  • 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.