Optimizing plant phosphoproteomic data independent acquisition: micro-pillar array columns and traditional packed bed technology

Significance of the Topic

Plant phosphorylation regulates critical processes such as signal transduction, stress responses and development
Deep and reliable profiling of phosphoproteomes is essential for advancing crop science and understanding molecular mechanisms
Combining high-throughput chromatography with data-independent acquisition (DIA) enables more comprehensive and reproducible analysis of low-abundance phosphopeptides

Study Objectives and Overview

The study compared short-gradient DIA performance of traditional packed-bed columns and microfabricated pillar array chromatography (µPAC) columns
Arabidopsis thaliana total protein and enriched phosphopeptide samples were analyzed to determine optimal column technology for plant phosphoproteomics
Four column formats were evaluated: two nanoViper PepMap Neo packed-bed columns (75 µm×15 cm, 150 µm×15 cm) and two µPAC Neo HPLC columns (50 cm and 5.5 cm high-throughput)

Methodology and Instrumentation

Sample Preparation

• Soil-grown Arabidopsis thaliana Col-0 rosettes harvested 21 days post-imbibition
• SDS-based SP3 bead extraction and tryptic digestion (Leutert et al. 2019; Mehta et al. 2022)
• Phosphopeptide enrichment using MagReSyn® Zi4+ IMAC HP particles on a Thermo Fisher KingFisher Apex

Chromatography and Mass Spectrometry

• Thermo Scientific Vanquish Neo UHPLC with trap-and-elute workflow and 15 µm EASY-Spray emitter
• NanoViper PepMap Neo columns (75 µm ID & 150 µm ID) at 1.5 µL/min; µPAC columns at 0.8–1.5 µL/min
• 15 min gradient from 4 % to 40 % ACN (0.1 % FA) with linear ramp to 99 % for column wash
• Orbitrap Astral MS in DIA mode: full scan at 240 k resolution, AGC 5e6; DIA windows covering m/z 380–980, AGC 5e4

Key Results and Discussion

Coverage and Identification

• Packed-bed 150 µm column (DNV150150PN) yielded highest phosphoprotein group IDs (3 518) and phosphopeptides (10 458)
• 5.5 cm µPAC high-throughput column achieved similar performance (2 925 proteins, 8 413 peptides) with fastest overhead

Peak Shape and Sensitivity

• Packed-bed columns produced sharper peaks and lower asymmetry for high-intensity peptides
• µPAC columns delivered higher peak heights and sensitivity for low-abundance phosphopeptides near quantification limits

Benefits and Practical Applications of the Method

Short gradients and high throughput are well suited for reproducible DIA workflows in plant phosphoproteomics
Choice of column can be tailored: packed-bed for maximum coverage and peak shape; µPAC for enhanced sensitivity of low-level targets
Implementation on Orbitrap Astral ensures high resolution and fast acquisition, enabling deep phosphoproteome profiling in under 20 minutes

Future Trends and Applications

Integration of longer and narrower µPAC formats for increased peak capacity
Development of real-time data processing and adaptive DIA window selection
Application of microfabricated columns in single-cell and spatial phosphoproteomics
Expansion to other post-translational modifications and challenging matrices

Conclusion

This comparative study highlights that traditional packed-bed technology provides robust coverage and peak quality, while µPAC pillars offer enhanced sensitivity for low abundance peptides
Researchers can select column format based on specific analytical goals, balancing throughput, coverage and sensitivity
The optimized DIA workflow on Orbitrap Astral represents a powerful approach for routine plant phosphoproteomics

References

Leutert B, et al. Efficient SP3 bead-based extraction and phosphopeptide enrichment in plant tissues. Methods Mol Biol. 2019;
Mehta B, et al. Enhanced plant phosphoprotein enrichment using MagReSyn® Zi4+ IMAC HP. J Proteome Res. 2022;
Dufresne CP, Talasila M, Larsen B, Uhrig RG. Optimizing plant phosphoproteomic data independent acquisition: micro-pillar array columns and traditional packed bed technology. Thermo Fisher Scientific Application Note. 2025.