iTRAQ Proteomics Profiling of Regulatory Proteins During Oligodendrocyte Differentiation

Recent evolution in proteomics approaches from two-dimensional gel electrophoresis to peptide-based “shotgun proteomics” methods has greatly enhanced the abilities of scientists to uncover expression changes among “low abundant” proteins. Shotgun proteomics methods typically employ stable isotope labeling techniques to distinguish peptides from the various sources that are compared. Recently, a new shotgun quantitative proteomics technology called i sobaric t ags for r elative and a bsolute q uantification (iTRAQ) has been developed for protein expression analysis. The major strength of the iTRAQ technology is its ability to compare the proteomic changes among multiple samples in a single experiment. Here we present a protocol on using the 8-plex iTRAQ approach for the discovery of molecular targets in oligodendrocyte progenitor cells during rapamycin-induced inhibition of differentiation. We provide the technical details on peptide labeling, chromatography, mass spectrometry, database search, and bioinformatics procedures for the identification of differentially expressed proteins.