Integrated Single-Tip IMAC-HILIC Enables Simultaneous 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, coanalysis of these PTMs is often complicated by their relatively
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 pipet tip, enabling concurrent enrichment
and sequential elution of phosphopeptides and N-glycopeptides. This
integrated workflow effectively reduces phosphopeptide coelution during
N-glycopeptide elution and streamlines sample processing. In direct
comparison with the tandem-tip TIMAHAC method, our single-tip strategy
achieves a comparable identification depth and offers superior quantitative
accuracy for N-glycopeptides. We further demonstrate its applicability
by examining the impact of calcium deprivation in Arabidopsis, revealing distinct global changes in both the phosphoproteome and
N-glycoproteome. Our optimized protocol thus provides a straightforward
and high-throughput platform for dual PTM profiling in complex plant
samples, paving the way for broader investigations of PTM crosstalk
in diverse physiological and stress responses.