Mass Spectrometric Analysis of Urinary N-Glycosylation Changes in Patients with Parkinson’s Disease

Abstract

Urine is thought to provide earlier and more sensitive molecular changes for biomarker discovery than blood. Numerous glycoproteins, peptides, and free glycans are present in urine through glomerular filtration of plasma, cell shedding, apoptosis, proteolytic cleavage, and exosome secretion. Urine biomarkers have enormous diagnostic potential, and the use of these biomarkers is a long-standing practice. The discovery of non-urological disease biomarkers from urine is also gaining attention due to its non-invasive sample collection and ease of analysis. Abnormal protein glycosylation in plasma or cerebrospinal fluid has been associated with Parkinson’s disease, however, whether urine with Parkinson’s disease has characteristic glycosylation remains to be explored. Here, we use mass spectrometry-based glycomics and glycoproteomics approaches to analyze urine samples for glycans, glycosites, and intact glycopeptides of urine samples. Reduced abundance of N-glycans was detected at the level of total glycans as well as specific glycosites of glycopeptides. The most abundant N-glycan in urine is S(6)­1H5N4F1; S(6)­2H5N4 and N4H4F1 are highly present in serum and urine, and 10 biantennary galactosylated N-glycans in the urine of PD patients were significantly decreased. The downregulation of sialylation may be due to the reduction of ST3GAL2. Site-specific N-glycosylation analysis revealed that AMBP, UMOD, and RNase1 have PD-specific N-glycosylation sites. GO and KEGG analysis revealed that N-glycosylation changes may provide clues to identify disease-specific glycosylation biomarkers in Parkinson’s disease.

Recent advances in N-glycan biomarker discovery among human diseases

“…In addition, Xu et al [170] observed elevated levels of glycans with core fucose, sialic acids, and bisecting GlcNAc in patients with PD. Intriguingly, in a followup study on urine samples, a decrease in the abundance of nearly all identified urine N-glycans, including those increased in serum samples, was noted in PD patients [171]. This finding suggested that alterations in the N-glycome may be distinct across different biological samples.…”

“…This finding suggested that alterations in the N-glycome may be distinct across different biological samples. In addition, specific changes in N-glycosylation of PD-associated glycoproteins, including ceruloplasmin and clusterin in serum, as well as 1-microglobulin/bikunin and uromodulin in urine, were identified [171]. In general, Nglycosylation plays a role in PD pathogenesis and is a potential disease-specific biomarker for PD.…”

Analysis of N‐ and O‐linked site‐specific glycosylation by ion mobility mass spectrometry: State of the art and future directions

“…While long activation times result in fragmentation in accordance with the bond dissociation energies, short activation times induce direct bond cleavage before the energy is redistributed over the entire molecule. Generally, the use of MS n /MS 2 acquisition with low energy can be and is often used for mapping the glycan site and possible glycan fragmentation and MS 3 for peptide backbone sequencing [83–85].…”