Protein glycosylation and phosphorylation are critical post-translational modifications (PTMs) governing nearly all cellular functions, yet their analysis remains challenging due to reliance on costly and unsustainable enrichment materials. Herein, we report a green synthesis of phosphorylated cellulose nanocrystals (P-CNCs) via one-step phosphoric acid hydrolysis, enabling dual-affinity enrichment of both glycopeptides and phosphopeptides. P-CNCs leverage abundant surface hydroxyl groups for hydrophilic interaction liquid chromatography (HILIC)-based glycopeptide capture, and intrinsic phosphate groups enable direct Ti4 + chelation (P-CNCs-Ti4+) for phosphopeptide enrichment without chemical derivatization. Using only 1 µL human serum, P-CNCs captured 2,025 N-linked and 2,183 O-GalNAcylated glycopeptides, including 10 previously unreported N-glycosylation chemical modifications. For phosphoproteomics, P-CNCs-Ti4 + enriched 5,225 phosphopeptides from mouse liver tissue, outperforming commercial TiO2 and identifying over 100 of 3-phosphoglyceryl modifications in glycolytic enzymes. Comprehensive life cycle assessment demonstrates the environmental sustainability of this approach, achieving a 76% cost reduction compared to commercial materials while significantly lowering the associated environmental footprint. This work pioneers P-CNCs as a sustainable, high-performance platform for multiplexed PTM profiling, bridging glycoproteomics and phosphoproteomics with broad applications in biomarker discovery and metabolic pathway analysis.
Li et al. (Fri,) studied this question.