Improved Method to Determine Protein Turnover Rates with Heavy Water Labeling by Mass Isotopomer Ratio Selection

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• Protein synthesis and degradation are crucial for gene expression.
• Measuring protein turnover rates is essential but analytically challenging.
• Existing methods for deuterium labeling mass spectrometry require refined analytical approaches.

Purpose of the Study:

• To develop an improved method for calculating protein synthesis rates from heavy water labeling mass spectrometry data.
• To enhance the accuracy and coverage of peptide analysis in protein turnover studies.
• To provide a robust computational workflow for proteome-wide protein kinetics.

Main Methods:

• A novel method combining nearest lookup of peptide isotope envelopes and rule-based selection of mass isotopomer pairs.
• Validation using a calibration standard of unlabeled and labeled proteomes.
• Reanalysis of 17 proteome-wide turnover experiments across four mouse organs.

Main Results:

• The new method significantly increases the coverage of well-fitted peptides in protein turnover experiments by up to 58 ± 13%.
• Demonstrated improved accuracy in calculating the molar fraction of new peptide synthesis.
• The Riana software tool implements this workflow for practical application.

Conclusions:

• The developed method enhances the analysis of protein synthesis and degradation kinetics.
• This approach improves the reliability and scope of proteome-wide protein turnover studies.
• The Riana tool facilitates advanced research into protein dynamics in biological systems.