Session: Parallel session 1 - Health and biological sciences

Serum N-glycomics by MALDI-TOF/TOF mass spectrometry for the diagnosis of congenital disorders of glycosylation (CDG)

Objectives



Glycosylation is the most represented post-transitional modification of proteins. Congenital disorders of glycosylation (CDG) constitute a family of over 150 rare inborn errors of metabolism characterized by defective synthesis of glycan chains, typically associated to poorly specific multivisceral clinical phenotypes. This variability makes the diagnosis of these pathologies long and complex, thus often requiring an arsenal of techniques to reach a definitive diagnosis. One relevant analytical strategy is to study whole N-glycans of human serum/plasma by MALDI-TOF mass spectrometry.

 

Methods



Profiles of total serum N-glycans (starting from 5 μL) were obtained by MALDI-TOF MS following N-glycan cleavage by peptide N-glycosidase F (PNGase F), protein precipitation and glycan purification by solid-phase extraction. N-glycans were then permethylated to increase detection sensitivity by MALDI-TOF MS.

These analyses were performed using a MALDI-TOF/TOF instrument (UltraFlextreme from Bruker). The N-glycans were analysed in positive ion mode and using the reflectron mode to obtain a better resolution.

 

Conclusions



The implemented MALDI-TOF approach allowed the detection of almost 40 distinct N-glycan structures in the serum sample from a healthy subject, with biantennary bi-sialylated structures as the most abundant species accounting for 30% of the total N-glycan pool. Manual assignment of glycan sequences was first performed on MS data and structures then confirmed by MS/MS experiments, when possible. This method was then applied to serum samples from CDG patients for diagnostic purposes (e.g. SLC35A3-CDG, TMEM165-CDG, PGM1-CDG). Using this method, the discrimination of healthy individuals from patients was possible in most cases.

Noteworthy, our analytical strategy has been used to evaluate the efficiency of a combined D-Gal/Mn2+ therapy administered to a 2-month-old TMEM165-CDG patient with a novel homozygous variant.