TiO₂ with Tandem Fractionation (TAFT): An Approach for Rapid, Deep, Reproducible, and High-Throughput Phosphoproteome...

Area of Science:

• Proteomics
• Biochemistry
• Cellular Signaling

Background:

• Phosphoproteomic analysis is crucial for understanding cellular signaling.
• Traditional methods require extensive phosphopeptide enrichment and fractionation.
• Existing workflows are often time-consuming and labor-intensive.

Purpose of the Study:

• To develop a rapid, high-throughput phosphoproteomic workflow.
• To improve the depth and reproducibility of phosphopeptide identification and quantification.
• To facilitate the study of signaling networks and clinical applications.

Main Methods:

• Titanium dioxide (TiO2) enrichment combined with tandem high-pH reverse-phase (HpRP) fractionation (TAFT).
• Streamlined workflow completed in under 3 hours without laborious phosphopeptide preparation.
• Application to HeLa and HepG2.2.15 cell lines.

Main Results:

• Identification and quantification of over 14,000 unique phosphopeptides from 1 mg of protein in <4 hours of MS measurement.
• Characterization of 21,281 phosphosites across two cell lines with >91% selectivity.
• High quantitative reproducibility with an average Pearson correlation of 0.90 between biological replicates.

Conclusions:

• The TAFT approach offers a rapid, deep, and reproducible method for phosphoproteome analysis.
• This cost-effective workflow accelerates the investigation of signaling pathways.
• Facilitates advancements in biological systems research and clinical applications.