Dynamic Proteomics: In Vivo Proteome-Wide Measurement of Protein Kinetics Using Metabolic Labeling

Methods Enzymol. 2015;561:219-76. doi: 10.1016/bs.mie.2015.05.018. Epub 2015 Jul 17.


Control of biosynthetic and catabolic rates of polymers, including proteins, stands at the center of phenotype, physiologic adaptation, and disease pathogenesis. Advances in stable isotope-labeling concepts and mass spectrometric instrumentation now allow accurate in vivo measurement of protein synthesis and turnover rates, both for targeted proteins and for unbiased screening across the proteome. We describe here the underlying principles and operational protocols for measuring protein dynamics, focusing on metabolic labeling with (2)H2O (heavy water) combined with tandem mass spectrometric analysis of mass isotopomer abundances in trypsin-generated peptides. The core principles of combinatorial analysis (mass isotopomer distribution analysis or MIDA) are reviewed in detail, including practical advantages, limitations, and technical procedures to ensure optimal kinetic results. Technical factors include heavy water labeling protocols, optimal duration of labeling, clean up and simplification of sample matrices, accurate quantitation of mass isotopomer abundances in peptides, criteria for adequacy of mass spectrometric abundance measurements, and calculation algorithms. Some applications are described, including the noninvasive "virtual biopsy" strategy for measuring molecular flux rates in tissues through measurements in body fluids. In addition, application of heavy water labeling to measure flux lipidomics is noted. In summary, the combination of stable isotope labeling, particularly from (2)H2O, with tandem mass spectrometric analysis of mass isotopomer abundances in peptides, provides a powerful approach for characterizing the dynamics of proteins across the global proteome. Many applications in research and clinical medicine have been achieved and many others can be envisioned.

Keywords: (2)H(2)O; Half-life; Kinetics; MIDA; Mass isotopomer distribution analysis; Metabolic label; Protein turnover; Proteome dynamics; Proteomics.

Publication types

  • Review

MeSH terms

  • Animals
  • Deuterium Oxide
  • Humans
  • Isotope Labeling / methods*
  • Proteome / metabolism*
  • Proteomics / methods*
  • Tandem Mass Spectrometry / methods*


  • Proteome
  • Deuterium Oxide