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Glycosylation Profiling of α/β T Cell Receptor Constant Domains Expressed in Mammalian Cells

  • Kai Zhang
  • Stephen J. Demarest
  • Xiufeng Wu
  • Jonathan R. Fitchett
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1575)

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.

Key words

Glycoprofiling Glycosylation Glycopeptide N-Linked Mass spectrometry (MS) LC-MS/MS ESI-MS T Cell receptor (TCR) 

Notes

Acknowledgments

The authors thank Bryan E. Jones and Wolfgang Glaesner for their managerial support, Flora Huang for the purification of TCR proteins, and Jayd Hanna and Benjamin Gutierrez for assistance with transient transfection and 293F cell culture.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Kai Zhang
    • 1
  • Stephen J. Demarest
    • 1
  • Xiufeng Wu
    • 1
  • Jonathan R. Fitchett
    • 1
  1. 1.Eli Lilly Biotechnology CenterSan DiegoUSA

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