A Method for the Characterisation of N-Glycome from Human Brain Glycoproteins

Background: Parkinson’s Disease (PD) is a neurodegenerative disorder characterised by the death of dopaminergic neurons in the substantia nigra. However, the molecular signature of PD is still not fully understood and there is a lack of studies regarding the protein glycosylation patterns in the brain and PD. Little is known on the role of N-glycans in the brain but many of the congenital glycosylation disorders result in neurological abnormalities suggesting their importance[1]. Given that glycans may behave as regulators of protein stability and that PD is associated with the aggregation of aberrant proteins, an understanding of how the glycosylation affects the disease makes it highly attractive to explore in order to comprehend their role in the regulation of PD.

Aim: To optimise the isolation of N-glycans from the human striatum and nigra and quantify their relative proportions using ultra performance liquid chromatography (UPLC).

Methods: Brain tissue was homogenised using RIPA buffer and the proteins were encapsulated in an acrylamide gel. After reduction and alkylation of the proteins, PNGAseF was used to release the N-glycans and 2-Aminobenzamide to label them. The labelled glycans were purified and analysed on UPLC.  

Results: Preliminary data shows reproducible chromatographic profiles of 60 peaks, containing one or more glycans, for both striatum and nigra. 13 peaks are significantly different between these regions. Most of the detected glycans in the striatum are neutral (78% both in healthy and PD), however this percentage differs in the nigra (85% in healthy vs 78% in PD).

Conclusions: This study describes an UPLC-based method for N-glycan profiling of brain tissue glycoproteins with high reproducibility.