Sustainable Phosphorylated Cellulose Nanocrystals: A Dual‐Affinity Platform for High‐Efficiency Enrichment of Intact Glycopeptides and Phosphopeptides

ABSTRACT 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 Ti ^{4 +} chelation (P‐CNCs‐Ti ⁴⁺ ) 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‐Ti ^{4 +} enriched 5,225 phosphopeptides from mouse liver tissue, outperforming commercial TiO ₂ 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.