Table 1.

Overview of the techniques to study membrane receptor glycosylation: applications and limitations.

Tools for studying membrane receptor glycosylation
Method	Working principle	Applications	Limitations	References
Quantitative and qualitive measurement of receptor glycosylation
SDS-PAGE and glycan-cleaving enzymes	Separating glycoforms by differences in molecular weight, using cell lysates treated with or without glycan-cleaving enzymes.	Quick and easy way to observe different glycoforms and roughly assess localization: membrane vs. cytosol.	No detailed information on glycan structure. Glycoforms with small or no differences in molecular weight cannot be separated.	Freeze and Kranz, 2010
Lectin blot	Separating glycoforms by differences in molecular weight and staining with glycan binding lectins.	Analyze different glycoforms in cell lysates and provides insights regarding glycan composition.	Low binding affinity and specificity of lectins. No detailed information on glycan structure.	Cao et al., 2013
Chemoenzymatic labeling	Building a monosaccharide analog into the glycan chain to introduce a chemical modification compatible with click chemistry reactions.	Specific glycan structures can be labeled on live cells or cell lysates.	No detailed information on glycan structure or site occupancy. Labeling is not specific for the protein of interest.	Lopez Aguilar et al., 2017
Lectin and antibody labeling	Visualizing specific glycan structures by lectin or antibody staining.	Labeling specific glycan structures for microscopy or flow cytometry.	Low binding affinity and specificity of lectins. Labeling is not specific for the protein of interest.	Tommasone et al., 2019
In situ proximity ligation assay	Antibodies conjugated to oligonucleotides can only ligate and be visualized when the antibody targets are in close proximity.	Provides information on the location of glycoforms within the cell and on the cell membrane.	No detailed information on glycan structure. Distance between the detected entities can be up to 40 nm, sometimes complicating data interpretation.	Soderberg et al., 2006
Glycomics	Analyzing glycan structure with mass spectrometry-based techniques.	Provides detailed structural information on glycans released from their protein backbone.	No information on the location of the glycosylation site within the protein.	Lauc and Wuhrer, 2017
Glycoproteomics	Analyzing glycopeptides with mass spectrometry-based techniques.	Provides information on the location of the glycosylation site and the glycan structure as well as micro- and macro-heterogeneity.	Glycan fine structures cannot be dissected when using large scale glycopeptide analysis. Complicated data analysis creating many false positive identifications.	Lauc and Wuhrer, 2017; Yang et al., 2017; Narimatsu H. et al., 2018
FRET-based labeling of glycoproteins	Energy transfer from a donor fluorophore to an acceptor fluorophore can only occur when the antibody targets are in close proximity.	Provides information on the cellular location of glycoforms.	No detailed information on glycan structure and site-occupancy.	Lin et al., 2014
Strategies for manipulation of membrane receptor glycosylation
Metabolic inhibitors	Different strategies, examples are sugar analogs, oligosaccharyltransferase inhibitors or inhibitors of glycosyltransferases.	Inhibition of the glycosylation pathway resulting in loss, truncation or modification of glycans.	Unwanted side-effects. Effect not specific for protein of interest.	Wojtowicz et al., 2012
Knockdown or knockout of glycosyltransferases	RNA interference or CRISPR-Cas9 genome editing.	Inhibition of the glycosylation pathway resulting in loss, truncation or modification of glycans.	Need for easy-to-transfect cell-line. Effect not specific for protein of interest.	Stolfa et al., 2016; Narimatsu Y. et al., 2018
Site-directed mutagenesis	Changing the glycosylation consensus sequence by changing the amino acid sequence of the protein and therefore removing the glycosylation site.	Removing one or multiple glycan chains from a specific protein.	Need for easy-to-transfect cell-line. Amino-acid substitution can affect other protein properties besides glycan presence. Modification of glycans is not possible.	Barbosa et al., 1987; Bordo and Argos, 1991; Weber et al., 2004