Congenital Disorders of Glycosylation (CDG) are genetic diseases characterized by altered glycosylation of proteins, lipids and/or both. To discover disease-associated biomarkers, as well as to identify changing in N-glycomic profiles supporting diagnosis of congenital disorders of glycosylation, was applied a High Throughput (HT) method based on liquid chromatography (LC) coupled with electrospray ionization (ESI)-mass spectrometry (MS). Before LC-MS analysis, to enhance detection sensitivity, was used RapiFluor-MS (RFMS) that has been developed as a new method for a fast deglycosylation and labeling of N-glycans such as monoclonal antibodies (mAbs). The RFMS protocol was slightly modified extending enzymatic digestion times. RFMS labeled N-glycans were separated and analyzed by Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS. This strategy allowed us to analyze structural details of patients' serum released N-glycans and, in some cases, to differentiate either structural isomers or isomers differing in the linkage type. In this study, we analyzed N-glycans of serum proteins in some CDG patients (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199- CDG). Unusual hybrid N-glycan structures with ?-1,2 terminal mannose residue and abnormal oligomannose N- structures were found in serum N-glycosylation profile of MAN1B1-CDG. A similar occurrence was showed in IgG and serum N-glycosylation analysis of ALG12-CDG. Total serum and IgG N-glycome analysis of MOGS-CDG indicated a series of fucosylated and unfucosylated hybrid N-glycans and additional oligomannose species. TMEM199 serum analysis revealed increased amounts of hyposialylated and hypogalactosylated N-glycans as associated with this disease.
HILIC-UPLC-MS N-GLYCAN CHARACTERIZATION IN THE DIAGNOSIS OF CONGENITAL DISORDERS OF GLYCOSYLATION
Angela Messina;Angelo Palmigiano;Domenico Garozzo
2020
Abstract
Congenital Disorders of Glycosylation (CDG) are genetic diseases characterized by altered glycosylation of proteins, lipids and/or both. To discover disease-associated biomarkers, as well as to identify changing in N-glycomic profiles supporting diagnosis of congenital disorders of glycosylation, was applied a High Throughput (HT) method based on liquid chromatography (LC) coupled with electrospray ionization (ESI)-mass spectrometry (MS). Before LC-MS analysis, to enhance detection sensitivity, was used RapiFluor-MS (RFMS) that has been developed as a new method for a fast deglycosylation and labeling of N-glycans such as monoclonal antibodies (mAbs). The RFMS protocol was slightly modified extending enzymatic digestion times. RFMS labeled N-glycans were separated and analyzed by Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS. This strategy allowed us to analyze structural details of patients' serum released N-glycans and, in some cases, to differentiate either structural isomers or isomers differing in the linkage type. In this study, we analyzed N-glycans of serum proteins in some CDG patients (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199- CDG). Unusual hybrid N-glycan structures with ?-1,2 terminal mannose residue and abnormal oligomannose N- structures were found in serum N-glycosylation profile of MAN1B1-CDG. A similar occurrence was showed in IgG and serum N-glycosylation analysis of ALG12-CDG. Total serum and IgG N-glycome analysis of MOGS-CDG indicated a series of fucosylated and unfucosylated hybrid N-glycans and additional oligomannose species. TMEM199 serum analysis revealed increased amounts of hyposialylated and hypogalactosylated N-glycans as associated with this disease.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
