Protein turnover, is the balance between protein synthesis and protein degradation, as the old or potentially damaged peptides/proteins are enzymatically hydrolyzed and replaced by newly synthesized copies. In the fields of protein chemistry and proteomics, it’s become more and more important to determine the protein turnover rate in protein homeostasis. Currently there are several technical solutions to determine the turnover rates of specific proteins, including pulse-chase and bleach-chase methods. The latter is applicable to fluorescently tagged proteins, and the as an isotopic tracer technique, the former is more popular because of its ease of operation, especially when stable isotopes are applied.
Labeling of cellular proteins is metabolically achieved by placing cells in a medium containing all components necessary for growth of cells in culture, except for stable isotope labeled specific amino acids for quite a short while (Pulse), and then the cell would be cultured in conventional medium with native components. The SIL amino acids would be transported across the plasma membrane and incorporated into newly synthesized proteins, which display identical physicochemical properties except molecular weight. These mass differences can be detected by high resolution mass spectrometry (Chase). By monitoring the decreasing abundance of SIL proteins, the pulse-chase technique is highly adaptable for:
Exploring the life cycle of endogenous proteins,
Many early similar work for protein turnover determination was finished with radioactive isotopes for metabolic labeling. With the development of mass spectrometry for proteomics research, stable isotopes such as 2H, 13C, or 15N in SIL amino acids are utilized in recent studies on protein homeostasis. The stable isotope labeling is flexible and almost applicable to all living organisms.
Mass spectrometry has become the dominant technical solution for the study of protein turnover rate for its wide availability and high resolution. A range of methodologies has been established to measure protein turnover for the proteins you are interested in, based on stable isotope labeling and mass spectrometry. Our experienced tech staff and technicians are glad to discuss the inquiry with you, to select and modify the protocols, in order to provide the optimal procedures for your case at cost-effective rate.