Integrated Single-Tip IMAC-HILIC Enrichment Enables Simultaneous and Efficient Analysis of Plant Phosphoproteomics and N-glycoproteomics

Protein phosphorylation and N-glycosylation are key post-translational modifications (PTMs) in plants that regulate critical signaling processes. However, analyzing both PTMs simultaneously remains challenging due to low abundance and divergent enrichment requirements. Here, we present a single-tip IMAC-HILIC approach that integrates immobilized metal affinity chromatography (IMAC) and hydrophilic interaction chromatography (HILIC) materials within one pipette tip, enabling concurrent enrichment and sequential elution of phosphopeptides and N-glycopeptides. This workflow effectively reduces coelution of phosphopeptides during N-glycopeptide elution and significantly streamlines sample processing. Benchmarking against the tandem-tip TIMAHAC method demonstrates comparable identification depth and superior quantitative accuracy for N-glycopeptides. We further apply IMAC-HILIC to examine calcium deprivation in Arabidopsis, revealing distinct global changes in both phosphoproteome and N-glycoproteome. Our optimized protocol offers a practical, high-throughput solution for dual PTM profiling in complex plant samples, paving the way for broader investigations for PTM crosstalk in diverse physiological and stress responses.