Glycosylation Profiling of α/β T Cell Receptor Constant Domains Expressed in Mammalian Cells
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Updated: Mar 6, 2026
Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques
Published on: July 5, 2018
Kai Zhang1, Stephen J Demarest2, Xiufeng Wu2
1Eli Lilly Biotechnology Center, 10300 Campus Point Drive, San Diego, CA, 92121, USA. zhangkai@lilly.com.
Abstract:
Glycoprofiling recombinant proteins expressed and secreted from mammalian cells is key to understanding their interactions with glycoprotein receptors in vivo. Recently, recombinant T cell receptors (TCRs) are being considered as therapeutic moieties. Here we present a mass spectrometry based protocol with a "bottom up" approach to characterize glycosylation in recombinant fusion proteins with α/β TCR constant domains expressed in mammalian cells. The protocol focuses on using peptide mass mapping and mass spectrometry for N-linked glycan profiling, including analyses of site occupancy, glycan heterogeneity, and possible glycan compositions and structures.
Insights
This study details a mass spectrometry method for analyzing N-linked glycans on recombinant T cell receptors (TCRs) produced in mammalian cells. The protocol characterizes glycosylation site occupancy and heterogeneity for therapeutic protein development.
Area of Science:
- Biochemistry
- Proteomics
- Glycoscience
Background:
- Glycosylation of recombinant proteins is crucial for their biological function and interaction with receptors.
- Recombinant T cell receptors (TCRs) show therapeutic potential but require thorough characterization.
- Mammalian cell expression systems are commonly used for producing complex recombinant glycoproteins.
Purpose of the Study:
- To develop and present a mass spectrometry-based protocol for detailed glycosylation analysis of recombinant fusion proteins containing TCR domains.
- To characterize N-linked glycosylation, including site occupancy and glycan heterogeneity.
- To provide a method for understanding the structural and compositional aspects of glycans on therapeutic TCR candidates.
Main Methods:
- A "bottom up" mass spectrometry approach was employed for glycan profiling.
- Peptide mass mapping was utilized to identify glycosylation sites.
- Mass spectrometry was used to analyze glycan heterogeneity and composition.
Main Results:
- The protocol successfully characterized N-linked glycosylation in recombinant fusion proteins with TCR domains.
- Detailed analysis of glycan site occupancy and heterogeneity was achieved.
- The method provides insights into potential glycan structures present on the recombinant proteins.
Conclusions:
- The presented mass spectrometry protocol offers a robust method for comprehensive glycoprofiling of recombinant TCRs.
- This characterization is essential for advancing the development of TCR-based therapeutics.
- Understanding glycosylation is key to predicting in vivo behavior and efficacy of glycoprotein therapeutics.
