Proteome Birthdating: A Single-Sample Approach for Measuring Global Turnover Dynamics and "Protein Age"

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Proteins exhibit diverse half-lives, leading to a wide range of molecular ages within cells.
• The influence of protein age on cellular interactions remains poorly understood.
• Studying protein age distributions and turnover is crucial for understanding cellular dynamics.

Purpose of the Study:

• To introduce and provide a practical guide for the proteome birthdating technique.
• To enable the analysis of protein age distributions and turnover kinetics.
• To facilitate investigations into the role of protein age in cellular pathways and disease.

Main Methods:

• Proteome birthdating involves differential labeling of proteins based on their synthesis time using isotopically labeled precursors.
• The technique utilizes tandem mass spectrometry (LC-MS/MS) for analyzing age distributions.
• The workflow includes cell culture, isotopic labeling, protein extraction, enzymatic digestion, peptide cleanup, mass spectrometry, and data processing.

Main Results:

• Proteome birthdating provides a method to barcode the proteome with age-specific isotopic labels.
• Global protein turnover kinetics can be determined from single, sequentially labeled samples.
• Age distributions of specific protein subsets (e.g., ubiquitinated proteins) can be analyzed.

Conclusions:

• Proteome birthdating is a versatile tool for studying protein age and turnover.
• The technique allows for the investigation of age selectivity in protein properties, cellular pathways, and disease states.
• This methodology opens new avenues for exploring the functional significance of protein age in biology.