Table 3.

Novel detection methods for glycosylation

	Methods	Principle	Advantages	Limitations	Reference
Separation enrichment affinity technique	LAC	Isolation and enrichment of intact glycopeptides is achieved by selective recognition of monosaccharides or glycans with specific structures by lectins immobilized on agarose or magnetic beads.	Widely used for the enrichment of specific N- and O-glycoproteins and glycopeptides. Multilectin affinity chromatography allows for the simultaneous enrichment of more diverse protein glycosylation by mixing multiple lectins.	Lectin specificity needs to be determined and multiple binding produces inertia.	519–523
	Hydrazine chemical enrichment	Trapping of glycopeptides by formation of covalent hydrazone bonds between the aldehyde group generated by oxidation of the glycan chain and the amino group on the hydrazide resin. Removal of the N-glycosyl chain by the PNGase F enzyme or chemical methods.	Simplified protein glycosylation and effective localization of N-glycosylation sites at the peptide level.	Complicated experimental procedures, especially the loss of glycan chain information.	524
	HILIC	A broad-spectrum enrichment method for the separation and enrichment of intact glycopeptides by the difference in polarity between glycosylated and non-glycosylated peptides.	Preserves the structure of intact glycopeptides. Enriches all types of intact glycopeptides without bias. Suitable for analyzing compounds in complex systems. Does not require expensive ion-pairing reagents and can be easily coupled with MS.	Co-expression of glycopeptides and non-glycopeptides leads to lower enrichment specificity.	525–527
	MAC	Glycosylated peptides are negatively charged and hydrophilic, which are selectively enriched by electrostatic interactions with metals.	High stability and low non-specific binding. Wide range of pH values. Multiple metal ions can be tested on the same resin to determine which metal ion is best suited for purifying the target protein.	Cannot be used on proteins that do not have a metal affinity. Some amino acids are susceptible to metal-catalyzed oxidation reactions leading to protein damage.	528
	Boric acid chemical method	Enrichment of intact glycopeptides using boric acid to form reversible covalent bonds with monosaccharides.	Captures and releases intact glycopeptides without affecting the structure of the glycan chain.	Different affinities between boric acid and different glycan chains.	529–531
Methods for glycoprotein identification/glycosylation site determination	Enzymatic method for PNGase F	Catalyze the non-lysosomal hydrolysis of an N(4)-(acetyl-β-d-glucosaminyl) asparagine residue into N-acetyl-β-d-glucosaminyl-amine and a peptide containing an aspartate residue.	Simplifies analysis of N-glycosylation. Good reusability and thermal stability.	Incomplete deglycosylation may lead to biased results.	532,533
	NMR	Caused by the spin motion of the nucleus.	Binding affinity and epitope information can be obtained. Analyze the structure, conformation and dynamics of carbohydrates and their interactions with biomolecular receptors such as proteins. Regardless of molecular size and without isotopic labeling.	Severe overlap of signal peaks, difficult resolution and low sensitivity. Multidimensional NMR requires milligram samples.	466,534,535
	Raman imaging	Utilizes spectral and spatial information to generate images.	A powerful visualization method with high sensitivity and specificity, without staining, with spatial and spectral resolution. Ability to record vibrational spectra in multiple regions to map the spatial distribution of glycans in human tissues.	Unable to quantify glycosylation levels. Detection of low abundance molecules requires next generation vibrating labels with higher sensitivity	467,536
Other detection technologies	Glycosylation-related gene testing	Using molecular biology and informatics techniques, the gene sequence of a glycan residue or glycosyltransferase is analyzed to derive a differential expression profile.	Suitable for the study of the specific structure of the lack of glycan, simple operation.	Technical and instrumental requirements.	537

LAC lectin affinity chromatography, HILIC hydrophilic interaction liquid chromatography, MS mass spectrometry, MAC metal affinity chromatography, NMR nuclear magnetic resonance