Multiplexed quantitative phosphoproteomics of cell line and tissue samples

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• Post-translational modifications (PTMs) regulate cellular mechanisms, with protein phosphorylation being crucial and often dysregulated in diseases like cancer.
• Understanding phosphorylation networks requires deep quantitative mapping of the phosphoproteome across numerous samples.
• Multiplexed proteomics enhances throughput for mass spectrometry-based analyses, enabling complex biological system investigations.

Purpose of the Study:

• To present a detailed protocol for multiplexed proteomics to map phosphoproteomes in cell culture and tissue samples.
• To enable high-throughput, quantitative analysis of protein phosphorylation sites.
• To facilitate a deeper understanding of biological networks regulated by phosphorylation.

Main Methods:

• Utilized multiplexed proteomics with tandem mass tag (TMT) reagents for barcoding up to 11 samples.
• Employed phosphopeptide enrichment using TiO2 and phosphotyrosine antibody technology.
• Applied the MultiNotch MS3 method for high-accuracy, reproducible quantitative mass spectrometry, complemented by MS2 (CID/HCD) for maximal phosphopeptide quantification.

Main Results:

• The protocol enables the quantification of over 20,000 unique phosphoforms from human proteome samples.
• Achieved high accuracy and reproducibility in quantitative phosphoproteome mapping.
• Required less than 8 hours of mass spectrometry time per sample.

Conclusions:

• This detailed protocol provides a robust and efficient method for deep phosphoproteome profiling.
• The developed technique significantly advances the ability to study phosphorylation-driven biological networks.
• Facilitates research into diseases associated with phosphorylation dysregulation, such as cancer.