In Vivo Isotopic Labeling of Proteins for Quantitative Proteomics
This protocol was adapted from “The Use of Mass Spectrometry in Proteomics,” Chapter 8, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.INTRODUCTION
Stable isotope coding strategies are of immense value in determining protein concentration changes in cells and tissues triggered by regulatory stimuli (e.g., drugs and toxins) and disease (e.g., caused by mutational changes). Recognizing and identifying the small number of proteins whose expression levels differ as a consequence of disease or external stimuli are complicated by the complexity of biological extracts and the fact that protein post-translational modifications are numerous and can occur at many sites on a protein. One way of quantifying global protein expression patterns involves in vivo labeling. Using the following protocol, stable isotopes can be incorporated into metabolic products, and the relative difference between these products from cells grown in normal or isotope-enriched media can be readily quantified by MS analysis.
