Profiling Glycoproteins Enriched by Multinanoparticle Protein Corona

Biofluids, such as plasma and cerebrospinal fluid (CSF), present significant challenges for proteome and glycoproteome analysis due to their complex protein composition and wide dynamic range of protein abundances. Nanoparticle (NP)-based enrichment methods that rely on the formation of protein coronas to compress dynamic range have emerged as promising approaches to improve the proteomic sampling depth in biofluids, yet implications of these methods for glycoproteome characterization remain underexplored. In this study, we investigate enrichment via multinanoparticle protein corona (abbreviated here as 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 an automated commercial platform, we demonstrate that NP enrichment significantly enhances N-glycoproteome coverage, particularly in plasma, and increases 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 multinanoparticle protein corona enrichment 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 protein level enrichment with MNPPC and glycopeptide enrichment for enhancing the glycoproteome coverage of human biofluids and provides a valuable framework for studying glycoproteins in complex biological samples.