Quantitative Proteomic Analysis to Profile Dynamic Changes in the Spatial Distribution of Cellular Proteins

Organelle protein profiles have traditionally been analyzed by subcellular fractionation of a specific organelle followedby the identification of the protein components of specific fractions containing the target organelle(s) using mass spectrometry(MS). However, because of limited resolution of the available fractionation methods, it is often difficult to isolate andthus profile pure organelles. Furthermore, many proteins (e.g., secretory proteins) are often observed to dynamically shuttlebetween organelles. Therefore, the determination of their true cellular localization requires the concurrent analysis of multipleorganelles from the same cell lysate. Here, we report an integrated experimental approach that simultaneously profiles multipleorganelles. It is based on the subcellular fractionation of cell lysates by density gradient centrifugation, iTRAQ labeling,and MS analysis of the proteins in selected fractions and principal component analysis (PCA) of the resulting quantitativeproteomic data. Quantitative signature patterns of several organelles, including the ribosome, mitochondria, proteasome, lysosome,endoplasmic reticulum (ER), and Golgi apparatus, have been acquired from a single multiplexed proteomic assay using iTRAQreagents. Through comparison PCA, we compare organelle profiles from cells under different physiological conditions to investigatechanges in organelle profiles between control and perturbed samples. Such quantitative proteomics-based subcellular profilingmethods thus provide useful tools to dissect the organization of cellular proteins into functional units and to detect dynamicchanges in their protein composition.