A robust method for quantitative high-throughput analysis of proteomes by 18O labeling.

Area of Science:

• Proteomics
• Mass Spectrometry
• Biotechnology

Background:

• Mass spectrometry-based quantitative proteomics is crucial for biological and medical research.
• Developing universally applicable, high-throughput techniques, especially for oxygen-18 (18O) labeling, remains a challenge.
• Statistical validation of quantitative proteomics methods needs further investigation.

Purpose of the Study:

• To present an improved high-throughput quantitative proteomics method utilizing whole proteome concentration, in-gel digestion, and 18O-labeling.
• To demonstrate the compatibility of off-gel isoelectric focusing with 18O peptide labeling across all pH ranges.
• To validate the statistical robustness and practical utility of the developed protocol.

Main Methods:

• Whole proteome concentration via SDS-PAGE.
• Optimized in-gel digestion and peptide 18O-labeling.
• Off-gel isoelectric focusing followed by liquid chromatography-LIT-MS separation.

Main Results:

• The off-gel technique is fully compatible with 18O peptide labeling.
• A statistical model confirmed that partial digestion and methionine oxidation do not impact protein quantification.
• Variances at scan, peptide, and protein levels are stable and reproducible across diverse proteomes.
• The method successfully detected expression changes in activated Jurkat T-cells.

Conclusions:

• The developed protocol provides a general, high-throughput approach for quantitative proteomics using 18O-labeling.
• The method is supported by a validated statistical model for the null hypothesis.
• This work represents the first report of a general stable isotope labeling protocol tested with multiple samples and detailed statistical analysis.