Profiling glycoproteins enriched by surface-functionalized nanoparticles

Biofluids, such as plasma and cerebrospinal fluid (CSF), present significant challenges for proteome and glycoproteome analysis due to their complex protein composition and the wide dynamic range of protein abundances. Nanoparticle (NP)-based enrichment methods have emerged as promising approaches to improve the proteomic sampling depth in biofluids, yet implications of these methods for glycoproteome characterization remains underexplored. In this study, we investigate NP enrichment, with and without subsequent mixed-mode anion exchange (MAX) enrichment, for characterizing 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 N-glycopeptides from low abundant glycoproteins. These N-glycoproteome improvements are further enhanced when combined with MAX enrichment. For the O-glycoproteome, NP enrichment alone outperformed NP+MAX, suggesting that MAX may preferentially enrich N-glycopeptides. We also generated the first application of the Seer Proteograph XT platform to analyze CSF, another biofluid of interest for biomarker discovery, and found that NP enrichment increased proteome and glycoproteome 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.