Proteomics of rat liver Golgi complex: Minor proteins are identified through sequential fractionation

The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for p roteomic analysis of this organelle. Our approach to Golgi proteomics was t o first isolate and characterize the University of Colorado, intact organel le free of proteins in transit by use of tissue pretreated with cycloheximi de. Then the stacked Golgi fraction was fractionated into biochemically def ined subfractions: Triton X-114 insoluble, aqueous, and detergent phases. T he aqueous and Department of Molecular detergent phases were further fracti onated by anion-exchange column chromatography. In addition, radiolabeled c ytosol was incubated with stacked Golgi fractions containing proteins in tr ansit, and the proteins bound to the Golgi stacks in an energy-dependent ma nner were characterized. All fractions were analyzed by two-dimensional (2- D) gel electrophoresis and identification numbers were given to 588 unique 2-D spots. Tandem mass spectrometry was used to analyze 93 of the most abun dant 2-D spots taken from preparative Triton X-114 insoluble, aqueous and d etergent phase 2-D gels. Fifty-one known and 22 unknown proteins were ident ified. This study represents the first installment in the mammalian Golgi p roteome database. Our data suggest that cell fractionation followed by bioc hemical dissection of specific classes of molecules provides a significant advantage for the identification of low abundance proteins in organelles.