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Nanoscale Solid-Phase Isobaric Labeling for Multiplexed Quantitative Phosphoproteomics.

Kosuke Ogata1, Chia-Feng Tsai1, Yasushi Ishihama1,2

  • 1Department of Molecular & Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.

Journal of Proteome Research
|July 22, 2021
PubMed
Summary
This summary is machine-generated.

We developed a highly sensitive nanoscale phosphoproteomics workflow for quantifying phosphorylated proteins. This method significantly improves sensitivity for analyzing small peptide amounts, aiding in drug target discovery.

Keywords:
TMT labelingmultiplexed phosphoproteomicsphosphopeptidessolid-phase reactortitanium oxide chromatography

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Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Phosphoproteomics is crucial for understanding cellular signaling pathways.
  • Conventional methods for quantitative phosphoproteomics face sensitivity limitations, especially with limited sample amounts.
  • Multiplexed quantitative analysis using tandem mass tags (TMT) is powerful but requires optimization for phosphopeptides.

Purpose of the Study:

  • To establish a highly sensitive workflow for multiplexed quantitative phosphoproteomics.
  • To optimize TMT labeling for phosphopeptides within a nanoscale solid-phase reactor.
  • To enhance the sensitivity of phosphoproteomics analysis for limited sample quantities.

Main Methods:

  • Development of a nanoscale solid-phase tandem mass tag (TMT) labeling reactor.
  • Enrichment of phosphopeptides using titanium oxide chromatography.
  • Optimization of TMT labeling post-enrichment for microscale reactions.
  • Application of the workflow to analyze selumetinib-treated HeLa cell proteins.

Main Results:

  • Achieved a nearly 10-fold increase in sensitivity compared to conventional solution-based TMT protocols.
  • Demonstrated successful quantification of selumetinib-regulated phosphorylated sites on a proteome scale.
  • Established a robust workflow for analyzing tens to hundreds of nanograms of phosphopeptides.

Conclusions:

  • The developed nanoscale phosphoproteomics workflow offers significantly enhanced sensitivity for quantitative analysis.
  • This method enables deeper insights into signaling pathways and drug-regulated phosphorylation events.
  • The protocol is suitable for proteome-wide quantification of phosphorylation changes in limited biological samples.