Linking post-translational modifications and protein turnover by site-resolved protein turnover profiling

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Protein turnover is crucial for cellular function but its regulation by post-translational modifications (PTMs) remains understudied.
• Existing proteome-wide turnover studies often overlook the impact of PTMs.

Purpose of the Study:

• To develop and apply a method for measuring protein turnover at the site-specific level, considering PTMs.
• To investigate how different PTMs influence the stability and dynamics of proteins across the proteome.

Main Methods:

• Utilized stable isotope labeling and mass spectrometry for quantitative proteomic analysis.
• Measured the turnover rates of over 120,000 peptidoforms, including over 33,000 modified peptides (phosphorylated, acetylated, ubiquitinated).
• Developed site-resolved protein turnover (SPOT) profiling to analyze PTM-specific turnover.

Main Results:

• Disclosed global and site-specific differences in protein turnover correlated with the presence or absence of PTMs.
• Observed diverging turnover rates for modified versus unmodified peptides, suggesting PTMs impact protein stability.
• Provided examples of how turnover data can elucidate PTM functions and created an accessible online tool for data exploration.

Conclusions:

• SPOT profiling is a powerful methodology for understanding PTM-mediated regulation of protein stability and function.
• The findings highlight the importance of considering PTMs in proteome-wide turnover studies.
• This approach can guide future research into the functional roles of specific PTMs.