Glycoproteomics in Cerebrospinal Fluid Reveals Brain-Specific Glycosylation Changes

Abstract

The glycosylation of proteins plays an important role in neurological development and disease. Glycoproteomic studies on cerebrospinal fluid (CSF) are a valuable tool to gain insight into brain glycosylation and its changes in disease. However, it is important to consider that most proteins in CSFs originate from the blood and enter the CSF across the blood–CSF barrier, thus not reflecting the glycosylation status of the brain. Here, we apply a glycoproteomics method to human CSF, focusing on differences between brain- and blood-derived proteins. To facilitate the analysis of the glycan site occupancy, we refrain from glycopeptide enrichment. In healthy individuals, we describe the presence of heterogeneous brain-type N-glycans on prostaglandin H2-D isomerase alongside the dominant plasma-type N-glycans for proteins such as transferrin or haptoglobin, showing the tissue specificity of protein glycosylation. We apply our methodology to patients diagnosed with various genetic glycosylation disorders who have neurological impairments. In patients with severe glycosylation alterations, we observe that heavily truncated glycans and a complete loss of glycans are more pronounced in brain-derived proteins. We speculate that a similar effect can be observed in other neurological diseases where a focus on brain-derived proteins in the CSF could be similarly beneficial to gain insight into disease-related changes.

The N-glycosylation defect in Lec5 and Lec9 CHO cells is caused by absence of the DHRSX gene

“…4E). This suggested that, rather than N-glycans being absent altogether, in many cases mature glycans might be replaced by immature glycans, agreeing with previous glycoproteomic studies on human LLO biosynthesis enzyme deficiencies (24, 25).…”

Dysregulated proteome and N‐glycoproteome in ALG1‐deficient fibroblasts

“…Additionally, we incorporated a novel xeno -tetrasaccharide (NeuAc-Hex-HexNAc 2 ) into the database, as previous studies had already identified this tetrasaccharide in the serum and fibroblasts of ALG1-CDG patients through released N-glycan analysis [ 10 , 11 ]. In a recent investigation, this tetrasaccharide were also identified on several proteins in cerebrospinal fluid from patients with ALG1-CDG [ 24 ]. Our analysis revealed an upregulation in the expression of numerous glycopeptides containing these oligosaccharide structures in all patient-derived ALG1 deficient fibroblasts irrespective of the gene variants.…”

“…Nevertheless, the studies did not explore the occurrence of this oligosaccharide on other cellular proteins or the presence of other shorter oligosaccharides. However, in a recent study, this novel tetrasaccharide was detected on various proteins using glycoproteomics study in the cerebrospinal fluid samples from one ALG1-CDG patient [ 24 ]. We detected increased abundance of this novel tetrasaccharide and other shorter oligosaccharides including a trisaccharide (Hex-HexNAc 2 ), a disaccharide (HexNAc 2 ) and a monosaccharide (HexNAc) on several cellular proteins.…”

Maximizing Glycoproteomics Results through an Integrated Parallel Accumulation Serial Fragmentation Workflow

“…Glycosylation is the most common post-translational modification of human proteins. , Its implication in all crucial processes of cellular life has resulted in a growing interest to analyze glycosylation to answer biological and clinical questions. ,− However, studying protein glycosylation is challenging due to the inherent structural complexity caused by micro- and macroheterogeneity and the existence of various glycan isomers. − As a result, the field of glycoproteomics is lacking behind its bottom-up proteomics counterpart where mass spectrometry (MS) has proven to be a powerful tool for identifying many thousands of proteins in a single LC-MS/MS experiment. − These advancements were supported by well-established workflows for sample preparation, MS acquisition, and data interpretation for bottom-up proteomics. , In comparison, the holistic analysis of glycopeptides remains challenging due to the physicochemical properties of glycopeptides and immature technology that lacks standardized workflows. Hence, MS identification rates of glycopeptides from complex mixtures are much lower compared to peptide identifications in bottom-up experiments. , When compared to bottom-up proteomics, glycoproteomics approaches require a tailored approach that favors the detection of glycopeptides over peptides.…”