N-glycans are often studied to discover disease-associated biomarkers, as well as to identify changing in glycomic profiles supporting diagnosis of those disorders directly connected to impaired glycan biosynthesis of glycoconjugates, such as congenital disorders of glycosylation (CDG). Strategies based on liquid chromatography (LC) coupled preferentially to electrospray ionization (ESI) - mass spectrometry (MS) have emerged as powerful analytical methods for N-glycan identification and characterization. To enhance detection sensitivity, glycans are commonly functionalized with a tag prior to LC-MS analysis. Since the majority of the techniques used for glycan derivatization are notoriously time-consuming, some commercial analytical kits were developed to perform a rapid deglycosylation and labelling of N-glycans linked specifically to monoclonal antibodies (mAbs). Adopting one of these commercial kits, RapiFluor-MS (RFMS), through a slightly modified protocol, we performed the enzymatic release and the N-glycan labelling of total serum and single serum glycoproteins from selected CDG patients (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199-CDG). Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS separation of derivatized N-glycans allowed us to differentiate either structural isomers and isomers differing for the linkage type, as hybrid glycan structures accumulating in N-glycan profile of MAN1B1-CDG and ALG12-CDG. According to patient immunological phenotype, serum IgG analysis of the Glucosidase 1-deficient patient (MOGS-CDG) showed significant N-glycosylation differences compared to control as the occurrence of the accumulating Glc3-Man7-GlcNAc2 glycoform (in accordance with the molecular defect) and, moreover, a generalized increase of sialylation. Serum N-glycan profiling of TMEM199-CDG showed, aside an increased amount of undersialylated biantennary structures, also an overall increase of the triantennary N-glycan component. All these applications demonstrated that RFMS method coupled to HILIC-UPLC-ESI-MS, represents a sensitive high throughput approach for serum N-glycome analysis and a valuable option for glycan detection and separation particularly for isomeric species.
HILIC-MS based glycomics in the diagnosis of Congenital Disorders of Glycosylation
A Palmigiano;A Messina;R Barone;L Sturiale;D Garozzo
2019
Abstract
N-glycans are often studied to discover disease-associated biomarkers, as well as to identify changing in glycomic profiles supporting diagnosis of those disorders directly connected to impaired glycan biosynthesis of glycoconjugates, such as congenital disorders of glycosylation (CDG). Strategies based on liquid chromatography (LC) coupled preferentially to electrospray ionization (ESI) - mass spectrometry (MS) have emerged as powerful analytical methods for N-glycan identification and characterization. To enhance detection sensitivity, glycans are commonly functionalized with a tag prior to LC-MS analysis. Since the majority of the techniques used for glycan derivatization are notoriously time-consuming, some commercial analytical kits were developed to perform a rapid deglycosylation and labelling of N-glycans linked specifically to monoclonal antibodies (mAbs). Adopting one of these commercial kits, RapiFluor-MS (RFMS), through a slightly modified protocol, we performed the enzymatic release and the N-glycan labelling of total serum and single serum glycoproteins from selected CDG patients (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199-CDG). Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS separation of derivatized N-glycans allowed us to differentiate either structural isomers and isomers differing for the linkage type, as hybrid glycan structures accumulating in N-glycan profile of MAN1B1-CDG and ALG12-CDG. According to patient immunological phenotype, serum IgG analysis of the Glucosidase 1-deficient patient (MOGS-CDG) showed significant N-glycosylation differences compared to control as the occurrence of the accumulating Glc3-Man7-GlcNAc2 glycoform (in accordance with the molecular defect) and, moreover, a generalized increase of sialylation. Serum N-glycan profiling of TMEM199-CDG showed, aside an increased amount of undersialylated biantennary structures, also an overall increase of the triantennary N-glycan component. All these applications demonstrated that RFMS method coupled to HILIC-UPLC-ESI-MS, represents a sensitive high throughput approach for serum N-glycome analysis and a valuable option for glycan detection and separation particularly for isomeric species.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
