Integrated Single-Tip IMAC-HILIC Enables Simultaneous 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, 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.

Keywords: N-glycoproteomics; enrichment; hydrophilic interaction Chromatography; immobilized metal affinity chromatography; phosphoproteomics.

1

A single IMAC-HILIC tip serves as a unified platform for the simultaneous enrichment of phosphopeptides and N-glycopeptides. Contaminant removal and proteolysis are performed using an S-Trap microcolumn. Digested peptides are eluted with 80%ACN/1%TFA into the IMAC-HILIC tip via centrifugation. Within the tip, N-glycopeptides and phosphopeptides are captured and subsequently eluted sequentially using 1% AA and ammonium phosphate solution, respectively. The enriched peptides are analyzed using an Evosep system coupled to a timsTOF HT mass spectrometer in the 30 SPD DDA mode. Phosphoproteomics and N-glycoproteomics data are processed with SpectroMine and FragPipe, respectively.

2

Evaluation of organic acids in the HILIC elution buffer on phosphopeptide retention using Fe³⁺-IMAC. (A) Number of unique peptides, phosphopeptides, and N-glycopeptides identified in the first elution fraction using three different organic acids: AA, TFA, and FA. (B) Number of unique monophosphorylated and multiply phosphorylated peptides in the second elution with ammonium phosphate following the first elution with AA, TFA, and FA. (C) Accumulated XIC area of monophosphorylated peptides identified in the second elution, expressed relative to the total intensity for the AA condition (set to 100%). (D) Boxplot illustrating the distribution of the CVs for phosphopeptides identified in the second elution following the first elution with AA, TFA, and FA.

3

Assessment of the N-glycopeptide elution efficiency using three organic acids in HILIC. (A) Number of unique peptides, N-glycopeptides, and phosphopeptides identified after elution with AA, TFA, and FA. (B) Accumulated XIC area of identified N-glycopeptides for each organic acid, with the total intensity for TFA set to 100%.

4

Impact of coelution on peptide identification and reproducibility of the two IMAC-HILIC fractions. (A) Number of unique peptides, phosphopeptides, and N-glycopeptides identified in the GP fraction after its elution with three acids: AA, TFA, and FA. (B) Boxplot illustrating the distribution of CVs for N-glycopeptides identified within the GP fraction when eluted with AA, TFA, and FA. (C) Number of unique monophosphorylated and multiply phosphorylated peptides identified in the subsequent PP fraction following the initial GP fraction elution with AA, TFA, and FA. (D) Overlap analysis of identified N-glycopeptides (left) and phosphopeptides (right) between the GP and PP fractions when AA was utilized as the elution buffer for the GP fraction.

5

Benchmarking the quantitative performance of the IMAC-HILIC tip against the TIMAHAC approach. Comparison of the number and percentage of quantifiable (A) N-glycopeptides and (B) phosphopeptides with CVs below 10% and 20%. Peptides were considered quantifiable if they were identified in at least two out of three technical replicates. (C) Distributions of peptide length and glycan saccharides count for N-glycopeptides identified using each method. (D) Histogram showing the distribution of phosphopeptide lengths in both approaches.

6

Analysis of the impact of calcium deprivation on the Arabidopsis phosphoproteome and N-glycoproteome using IMAC-HILIC tip. (A) Comparison of relative protein abundance distributions in the global proteome for proteins identified in the PP and GP fractions. (B) Venn diagram showing the overlap of proteins identified in the global proteome, phosphoproteome, and N-glycoproteome fractions. (C) Heatmaps depicting hierarchical clustering of quantifiable phosphorylation sites and N-glycopeptides significantly altered by EGTA treatment. (D) Selected GO Biological Process terms enriched among phosphoproteins and N-glycoproteins that were significantly modulated under EGTA treatment.