HILIC-UPLC-MS for high throughput and isomeric N-glycan separation and characterization in Congenital Disorders Glycosylation and human diseases

N-glycan analyses may serve uncovering disease-associated biomarkers, as well as for profiling distinctive changes supportingdiagnosis of genetic disorders of glycan biosynthesis named congenital disorders of glycosylation (CDG). Strategies based onliquid chromatography (LC) preferentially coupled to electrospray ionization (ESI) - mass spectrometry (MS) have emerged aspowerful analytical methods for N-glycan identification and characterization. To enhance detection sensitivity, glycans arecommonly labelled with a functional tag prior to LC-MS analysis. Since most derivatization techniques are notoriously timeconsuming,some commercial analytical kits have been developed to speed up N-deglycosylation and N-glycan labelling ofglycoproteins of pharmaceutical and biological interest such as monoclonal antibodies (mAbs). We exploited the analyticalcapabilities of RapiFluor-MS (RFMS) to perform, by a slightly modified protocol, a detailed N-glycan characterization of totalserum and single serum glycoproteins from specific patients with CDG (MAN1B1-CDG, ALG12-CDG, MOGS-CDG,TMEM199-CDG). This strategy, accomplished by Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS separationof the RFMS derivatized N-glycans, allowed us to uncover structural details of patients serum released N-glycans, thus extendingthe current knowledge on glycan profiles in these individual glycosylation diseases. The applied methodology enabled todifferentiate in some cases either structural isomers and isomers differing in the linkage type. All the here reported applicationsdemonstrated that RFMS method, coupled to HILIC-UPLC-ESI-MS, represents a sensitive high throughput approach for serumN-glycome analysis and a valuable option for glycan detection and separation particularly for isomeric species.