TY - CHAP
T1 - Glycosylation Profiling of Glycoproteins Secreted from Cultured Cells Using Glycan Node Analysis and GC-MS
AU - Aguilar Díaz de león, Jesús S.
AU - Borges, Chad R.
N1 - Publisher Copyright:
© 2021, Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2021
Y1 - 2021
N2 - Glycan “node” analysis is the process by which pooled glycans within complex biological samples are chemically deconstructed in a way that facilitates the analytical quantification of uniquely linked monosaccharide units (glycan “nodes”). It is based on glycan methylation analysis (a.k.a. linkage analysis) that has historically been applied to pre-isolated glycans. Thus, when using glycan node analysis, unique glycan features within whole biospecimens such as “core fucosylation,” “α2-6 sialylation,” “β1-6 branching,” “β1-4 branching,” and “bisecting GlcNAc,” are captured as single analytical signals by GC-MS. Here we describe the use of this methodology in cell culture supernatant and in the analysis of IgG (alpha-1 antitrypsin) glycans. The effect of IL-6 and IL-1β cytokines on secreted hepatocyte protein glycan features is demonstrated; likewise, the impact of neuraminidase treatment of IgG is illustrated. For the majority of glycan nodes, the assay is consistent and reproducible on a day-to-day basis; because of this, relatively subtle shifts in the relative abundance of glycan features can be captured using this approach.
AB - Glycan “node” analysis is the process by which pooled glycans within complex biological samples are chemically deconstructed in a way that facilitates the analytical quantification of uniquely linked monosaccharide units (glycan “nodes”). It is based on glycan methylation analysis (a.k.a. linkage analysis) that has historically been applied to pre-isolated glycans. Thus, when using glycan node analysis, unique glycan features within whole biospecimens such as “core fucosylation,” “α2-6 sialylation,” “β1-6 branching,” “β1-4 branching,” and “bisecting GlcNAc,” are captured as single analytical signals by GC-MS. Here we describe the use of this methodology in cell culture supernatant and in the analysis of IgG (alpha-1 antitrypsin) glycans. The effect of IL-6 and IL-1β cytokines on secreted hepatocyte protein glycan features is demonstrated; likewise, the impact of neuraminidase treatment of IgG is illustrated. For the majority of glycan nodes, the assay is consistent and reproducible on a day-to-day basis; because of this, relatively subtle shifts in the relative abundance of glycan features can be captured using this approach.
KW - Aberrant Glycosylation
KW - Antibody Glycosylation Profiling
KW - Cell Culture Supernatant
KW - GC-MS
KW - Glycan Nodes
KW - Glycan Permethylation
KW - Glycans
KW - Glycosylation Profiling
KW - Secreted Glycoproteins
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U2 - 10.1007/978-1-0716-1241-5_22
DO - 10.1007/978-1-0716-1241-5_22
M3 - Chapter
C2 - 33908017
AN - SCOPUS:85105068200
T3 - Methods in Molecular Biology
SP - 317
EP - 330
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -