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

ABSTRACT: 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.

INSTRUMENT(S): timsTOF HT

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Culture

SUBMITTER: Ting-An Chen

LAB HEAD: Chuan-Chih Hsu

PROVIDER: PXD061247 | Pride | 2025-05-14

REPOSITORIES: Pride

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			Action	DRS
	1132390_1AA-1-E.zip	Other
	1132392_1AA-3-E.zip	Other
	1132393_1AA-4-E.zip	Other
	1132394_0p5TFA-1-E.zip	Other
	1132395_0p5TFA-2-E.zip	Other

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