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Related Experiment Video

Updated: Jul 4, 2026

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues
09:16

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.

Biorxiv : the Preprint Server for Biology
|July 3, 2026
PubMed
Summary

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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).
Keywords:
data-independent acquisition (DIA)dia-PASEFhigh-throughput proteomicsphosphoproteomicsretention time summationsynchro-PASEFtimsTOF

More Related Videos

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae
15:41

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Published on: October 12, 2009

Related Experiment Videos

Last Updated: Jul 4, 2026

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues
09:16

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Published on: May 4, 2022

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae
15:41

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Published on: October 12, 2009

  • 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.