Table 2.

Analysis/resolution of glycoprotein microheterogeneity using other techniques in conjunction with lectin affinity chromatography

Serial no.	Origin and nature of glycoprotein microheterogeneity	Technique(s) used for heterogeneity resolution	Applications/analysis/conclusions/remarks	Reference
1	α1-antichymotrypsin microheterogeneity	immunoaffinoelectrophoresis with free Concanavalin A (Con A) in the first dimension; Con A Sepharose Affinity Chromatography (Con A-SAC) and high resolution 1H-NMR spectroscopy	Con A-SAC separates the protein into 3 fractions: Con A-non reactive form (with 4 triantennary glycans), a Con A weakly reactive form (with 3 triantennary and 1 diantennary glycans) and a Con A reactive form (with 1 triantennary and 3 diantennary glycans). There is an increased proportion of Con A non-reactive form in patients developing a systemic disease (systemic lupus erythematosus, rheumatoid arthritis, temporal arteritis).	[37]
2	Glycosylation status of serum transferrin as a biochemical index of carbohydrate deficient glycoprotein syndrome type I	Capillary zone electrophoresis and a novel HPLC strategy for quantification of glycans released by exoglycosidase treatment	Hexa-, penta-, and tetrasialoforms of human serum transferrin are present in both normal and carbohydrate-deficient glycoprotein syndrome type I serum samples. In addition, the carbohydrate deficient glycoprotein syndrome type I transferrin also contained a disialoform, representing a glycoform in which one of the two N-glycosylation sites is unoccupied, and non-glycosylated form where both remain unoccupied. This could be used as a rapid diagnostic test for the carbohydrate-deficient glycoprotein syndromes group of diseases.	[38]
3	Fel d1 (cat allergen 1)	HPLC and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)	The allergen is a 38-kDa dimer of two 19 kDa subunits, each of which comprises a light α-chain and a heavy β-chain containing an N-linked oligosaccharide on Asn33; Fel d1 is found to be partially truncated and to exist in several isoforms; the glycan is a heterogeneous triantennary complex type structure; and the heterogeneity is caused by terminal sialic acid and a fucose residue attached to a β-galactose residue.	[39]
4	Transferrin, α1antitrypsin, haptoglobin β-chain, and α1-acid glycoprotein microheterogeneity in serum and liver of patients with carbohydrate-deficient glycoprotein syndrome type I	High-resolution two-dimensional electrophoresis (2-DE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)	Serum glycoproteins in all patients showed a cathodal shift and decreased mass. The two-dimensional pattern of immunodetected precursors of serum proteins in liver cells from patients with CDGS showed abnormal low-mass precursors and absence precursors normally found in controls. These results suggest that these abnormal precursors accumulate during early oligossaccharide processing of the nascent protein-bound oligosaccharides and that glycoprotein precursors undergo an altered intracellular transport while the post-translational processing along the normal pathway is still apparently functioning in patients with CDGS.	[40]
5	Glycoform heterogeneity of follicle stimulating hormone (FSH) and luteinizing hormone (LH) through the normal menstrual cycle and in the post-menopausal state	Con A-SAC	The changes in gonadotropin glycoforms occur through the menstrual cycle which are related to changes in the prevailing steroid environment. Following the menopause oestrogenic loss resulted in acidic, relatively, simple glycoforms.	[41]
6	Sugar sequence and branch structure of the oligosaccharides in RNase B	GCC-LC/MS in the positive ion mode and (LC/MS/MS)	Identification of 1 Man5GlcNAc, 3 Man6GlcNAc, 3 Man7GlcNAc, 3 Man8GlcNac, 1 Man9GlcNac, and an oligosaccharide having six hexose units (Hex) and 2 N-acetylhexosamine units (HexNAc). These techniques can be used for elucidation of the distribution of oligosaccharides too.	[42]
7	Armadillidium vulgare androgenic hormone glycoforms (AH1 and AH2)	HPLC and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)	Amino acid (aa) sequence of the two chains A (29-aa long) and B (44-aa long) of the two glycoforms are identical; Asn18 of chain A is N-glycosylated.	[43]
8	Glycosylation sites and preliminary glycosylation pattern in erythropoietin (EPO) and the detailed site-specific carbohydrate heterogeneity	Liquid chromatography (LC) mass spectrometry (MS) with graphitized carbon column (GCC), coupled with tandem mass spectrometry (LC/MS/MS)	The di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyl lactosamines, are combined with Asn24.	[44]
9	Glycosylation pattern of human epidermal growth factor receptor (EGFR)	Con A-SAC, anion exchange chromatography, HPLC and high pH anion-exchange chromatography; NMR spectroscopy and mass spectrometry	32 new complex-type glycans are characterized Oligomannose-type glycans range from Man5GlcNAc2 to Man8GlcNAc2. Di-, tri′- and tetraantennary complex-type structures are present, both neutral and (alpha2–3)-sialylated (up to tetrasialo), comprising 24% and 59%, respectively, of the total carbohydrate moiety.	[45]
10	Microheterogeneity of the IgA1 hinge glycopeptide (HGP33) having multiple O-linked oligosaccharides	Jacalin affinity chromatography and capillary electrophoresis	The self-aggregation of IgA1 is closely connected with the glycoform of a mucin-type sugar chain on its hinge portion (HGP33). Jacalin affinity chromatography separated the normal human serum IgA1 into two subfractions as: the monomeric form (eluted by 0.25 mM galactose and abundant in the sialic acid-rich components) and the aggregated form (eluted by 0.8 mM galactose, and abundant in the sialic acid-poor components). Application of CE analysis to HGP33 indicated that the monomeric IgA1 was composed of a relatively complete molecule with respect to the glycoform rather than the aggregated IgA1.	[46]