[Chuan-Chih Hsu] Integrated Single-Tip IMAC-HILIC Enables Simultaneous Analysis of Plant Phosphoproteomics and N-Glycoproteomics
POST:
A single IMAC-HILIC tip serves as a unified platform for the simultaneous enrichment and sequential elution of plant phosphopeptides and N-glycopeptides
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 both phosphopeptides and 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.
Chin-Wen Chen, Assistant R&D Scientist at the Proteomics Core Laboratory, and Ting-An Chen, full-time Research Assistant, contributed equally to this work as co-first authors. This study was published in the Journal of Proteome Research, a journal of the American Chemical Society (ACS).