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

Glycoconj J. 2021 Apr;38(2):201-211. doi: 10.1007/s10719-020-09947-7. Epub 2020 Sep 11.


N-glycan analyses may serve uncovering disease-associated biomarkers, as well as for profiling distinctive changes supporting diagnosis of genetic disorders of glycan biosynthesis named congenital disorders of glycosylation (CDG). Strategies based on liquid chromatography (LC) preferentially coupled 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 labelled with a functional tag prior to LC-MS analysis. Since most derivatization techniques are notoriously time-consuming, some commercial analytical kits have been developed to speed up N-deglycosylation and N-glycan labelling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies (mAbs). We exploited the analytical capabilities of RapiFluor-MS (RFMS) to perform, by a slightly modified protocol, a detailed N-glycan characterization of total serum 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 separation of the RFMS derivatized N-glycans, allowed us to uncover structural details of patients serum released N-glycans, thus extending the current knowledge on glycan profiles in these individual glycosylation diseases. The applied methodology enabled to differentiate in some cases either structural isomers and isomers differing in the linkage type. All the here reported 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.

Keywords: CDG; HILIC; High throughput N-glycomics; LC-MS; Oligomannose type N-glycans.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Chemical Analysis / methods
  • Chromatography, High Pressure Liquid / methods
  • Congenital Disorders of Glycosylation / blood*
  • Humans
  • Isomerism
  • Mannosidases / deficiency
  • Membrane Proteins / deficiency
  • Polysaccharides / blood*
  • Polysaccharides / chemistry*
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • alpha-Glucosidases / metabolism


  • Membrane Proteins
  • Polysaccharides
  • TMEM199 protein, human
  • MAN1B1 protein, human
  • Mannosidases
  • glucosidase I
  • alpha-Glucosidases