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Updated: Jul 4, 2026
A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues
Published on: May 4, 2022
Systematic optimization and benchmarking of synchro-PASEF for high-throughput phosphoproteome profiling
Dain R Brademan1, Angelina A Mullarkey1, Mia L Greeson1
1Department of Molecular & Cellular Physiology, Stanford School of Medicine, Stanford, CA, USA.
This study benchmarks synchro-Parallel Accumulation-Serial Fragmentation (synchro-PASEF) for phosphoproteomics, finding it offers comparable biological insights to data-independent acquisition-Parallel Accumulation-Serial Fragmentation (dia-PASEF) with optimized methods and retention time summation. The research provides a framework for high-throughput phosphoproteomics.
Area of Science:
- Proteomics
- Mass Spectrometry
- Systems Biology
Background:
- High-throughput data-independent acquisition (DIA) with short chromatography is key for systems biology and clinical proteomics.
- Rapid separations require faster mass spectrometer cycle times for quantitative depth and reproducibility.
- The synchro-PASEF mode offers efficient ion sampling with shortened cycle times, but its phosphoproteomics performance is unevaluated.
Purpose of the Study:
- To systematically optimize and benchmark synchro-PASEF for phosphoproteomics.
- To compare synchro-PASEF performance against dia-PASEF across different chromatographic separations.
- To evaluate the utility of retention time summation (RTsum) for enhancing phosphoproteomics data.
Main Methods:
- Systematic optimization of synchro-PASEF parameters (window number, isolation width, gradient length).
- Benchmarking against two dia-PASEF methods using sub-hour separations on timsTOF mass spectrometers.
- Application of retention time summation (RTsum) for data analysis.
- Modeling phosphoproteomic dose-response using β2-adrenergic receptor activation.
Main Results:
- Optimized synchro-PASEF quantified over 19,000 phosphosites in a 23-minute separation.
- RTsum increased phosphopeptide identifications by 5-20% and reduced phosphosite CVs by up to 30%.
- Both synchro-PASEF and dia-PASEF demonstrated concordant phosphoproteomic responses and predicted PKA substrates.
Conclusions:
- Synchro-PASEF provides comparable biological performance to dia-PASEF for high-throughput phosphoproteomics.
- Method optimization is critical for synchro-PASEF performance, balancing window parameters and gradient length.
- RTsum is a valuable post-acquisition tool for improving phosphoproteomics data quality and identification depth.
