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Profiling glycoproteins enriched by surface functionalised nanoparticles

ABSTRACT: Biologically relevant biofluids, such as plasma and cerebrospinal fluid (CSF), present significant challenges for glycoproteomic analysis due to their complex protein composition and the wide dynamic range of protein abundances. Nanoparticle-based (NP) enrichment has previously shown promise in enhancing the detection of low-abundance proteins, yet the glycoproteome has been underexplored. In this study, we investigate the use of NP enrichment with glycopeptide enrichment, in the form of a mixed-mode anion exchange chromatography, to characterize N- and O-linked glycopeptides in human plasma and CSF. Using the Seer Proteograph XT platform, we demonstrate that NP enrichment significantly enhances the N-glycoproteome coverage, particularly in plasma, and increases the detection of low abundant N-glycopeptides. When combined with MAX enrichment, a further improvement in glycopeptide detection was observed. For the O-glycoproteome, NP enrichment alone outperformed NP+MAX, suggesting that MAX may favor N-glycopeptides. We also explored CSF, a biofluid of interest for biomarker discovery, and found that NP enrichment increased proteome depth, with notable identification of extracellular vesicle protein biomarkers. Overall, this study highlights the synergy of NP-based enrichment and glycopeptide enrichment for enhancing the glycoproteome coverage of human biofluids and provides a valuable framework for studying glycoproteins in complex biological samples.

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ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Emmajay Sutherland

LAB HEAD: Nicholas M. Riley

PROVIDER: PXD059675 | Pride | 2026-01-09

REPOSITORIES: Pride

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Project description:A large family of GalNAc transferases (GalNAc-Ts) catalyzes the covalent attachment of N-Acetylgalactosamine (GalNAc) to serine and threonine residues on proteins that pass through the secretory pathway in the first committed step of mucin-type O-glycosylation. Abnormalities in the activity of individual GalNAc-Ts can result in congenital disorders of O-glycosylation (CDG) and influence other biological functions. Although ~85% of proteins that pass through the secretory pathway are modified with O-glycans, only 47 O-glycosylation sites (O-glycosites) from 22 mouse glycoproteins and 17 publications are present on UniProt and an O-glycoprotein database (http://www.oglyp.org/). A compilation of all in vivo O-glycosites (the O-glycoproteome) would be an invaluable step in determining the function of proteins decorated with N-Acetylgalactosamine and what role(s), if any, the O-glycans play. While approaches to delineate O-glycosites have been developed for simple, genetically engineered cell lines, there is no universal approach to mapping the O-glycosites of glycoproteins in complex tissue extracts or biological fluids. We have developed chemical and enzymatic conditions that cleave solitary O-glycans while leaving the modified core protein/peptides assayable by mass spectrometry (MS). The methodology permits the mapping of thousands of O-glycosites decorated with Tn or T antigen from tissues or biological fluids. We then integrated an HCD-pd-EThcD MS workflow and software, including MSFragger-Glyco, pGlyco3, and O-Pair, to study the mouse brain, heart, lung, liver, spleen, kidney, colon, muscle, submandibular gland, and whole blood. The integrated approach identified 2154 solitary O-glycosites from 595 glycoproteins. The O-glycosites and glycoproteins displayed consensus motifs and Gene Ontology (GO) terms for O-glycoproteins. Limited overlap of O-glycosites was observed with protein O-GlcNAcylation and phosphorylation sites. Integrating quantitative glycoproteomics and proteomics revealed a tissue-specific regulation of O-glycosites that the differential expression of Galnt isoenzymes in tissues partly contributes to. We next used established quantitative glycoproteomics and proteomics methods to identify site-specific substrates that may contribute to biologically relevant phenotypes when Galnt2 was genetically ablated in a Galnt2-null mouse model, which presents with lipid and metabolic dysregulation. Our findings suggest networks of Galnt2 direct and indirect interactions that may explain the complex metabolic phenotypes. The mouse O-glycoproteome-map and quantitative glycoproteomics/proteomics approach will provide a valuable foundation for revealing the significance of O-glycosylation biology in CDGs and other diseases and conditions.

			Action	DRS
	CSF-DIA-DB.fasta	Fasta
	CSF_DIANN_GFP_report.tsv	Tabular
	CSF_DIANN_report.tsv	Tabular
	CSF_DIA_GFP_400-500.raw	Raw
	CSF_DIA_GFP_500-600.raw	Raw

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Profiling glycoproteins enriched by surface functionalised nanoparticles

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