Profiling glycoproteins enriched by surface functionalised nanoparticles

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.