Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis

Area of Science:

• Proteomics
• Biochemistry
• Mass Spectrometry

Background:

• Global protein phosphorylation analysis using mass spectrometry is crucial for biological and biomedical research.
• Studying the phosphoproteome presents challenges due to low stoichiometry and the need for residue-specific data, impacting reproducibility and coverage.
• Optimizing phosphoproteomic workflows requires careful attention to sample preparation, data acquisition, and processing.

Purpose of the Study:

• To evaluate the impact of various factors on the performance of proteome-wide phosphoprotein analysis protocols.
• To establish a standardized reference sample for benchmarking phosphoproteomics strategies.
• To assess inter-laboratory reproducibility and performance in phosphoproteomic studies.

Main Methods:

• The Proteomics Multicentric Experiment 11 (PME11) involved distributing a reference sample (yeast extract spiked with a phosphomix standard) to 31 laboratories worldwide.
• Thirty-six datasets from 23 participating laboratories were analyzed.
• Performance was evaluated based on reproducibility, qualitative and quantitative robustness, and phosphoproteome coverage.

Main Results:

• The PME11 reference sample proved suitable for benchmarking and optimizing phosphoproteomics strategies.
• Analysis revealed the influence of different experimental variables on phosphoproteomic workflow performance.
• The study provided a basis for ranking intra- and inter-laboratory performance in phosphoproteomics.

Conclusions:

• The PME11 experiment successfully validated a reference sample for standardizing and improving phosphoproteomic analyses.
• This multicentric approach highlighted key factors affecting phosphoproteomics outcomes and inter-laboratory variability.
• The findings support the optimization of phosphoproteomic workflows for more robust and reproducible results in biological research.