title: Glycosylation Profiling of α/β T Cell Receptor Constant Domains Expressed in Mammalian Cells.
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.
authors.affiliation: Eli Lilly Biotechnology Center, 10300 Campus Point Drive, San Diego, CA, 92121, USA. zhangkai@lilly.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Eli Lilly Biotechnology Center, 10300 Campus Point Drive, San Diego, CA, 92121, USA.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Demarest
authors.affiliation: Eli Lilly Biotechnology Center, 10300 Campus Point Drive, San Diego, CA, 92121, USA.
authors.forename: Xiufeng
authors.initials: X
authors.lastname: Wu
authors.affiliation: Eli Lilly Biotechnology Center, 10300 Campus Point Drive, San Diego, CA, 92121, USA.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Fitchett
mesh_terms: D002853:Chromatography, Liquid; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D010449:Peptide Mapping / Q000379:methods*; D000072417:Protein Domains; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins
keywords: ESI-MS; Glycopeptide; Glycoprofiling; Glycosylation; LC-MS/MS; Mass spectrometry (MS); N-Linked; T Cell receptor (TCR)