Identifying Bona Fide Components of an Organelle by Isotope-Coded Labeling of Subcellular Fractions: An Example in Peroxisomes

Organelles are biochemically distinct subcellular compartments that perform specific functions within a cell. These roles are regulated by the complement of proteins associated with each organelle. Thus, a comprehensive understanding of an organelle’s proteome is necessary to elucidate the diverse roles of each organelle. Mass spectrometry combined with biochemical fractionation methods has enabled the proteomic characterization of several organelles. However, due to the poorly quantitative nature of mass spectrometry and the inability to generate pure fractions of an organelle, distinguishing bona fide components of the organelle from contaminants of the fraction is a significant challenge. We have addressed this limitation by adopting quantitative mass spectrometric approaches to identify proteins that enrich in a purified fraction of organelles relative to a crude or contaminating fraction. The methods for the analyses of the yeast peroxisome are described in detail; however, these concepts are generally applicable to the study of other organelles.