Comparison of Golgi apparatus and endoplasmic reticulum proteins from livers of juvenile and aged rats using a novel technique for separation and enrichment of organelles.
The broad dynamic range of protein abundances, which can vary from about 10(6) for cells to 10(10) for tissues in complex proteomes, continues to challenge proteomics research. Proteome analysis, in particular organelle proteomics, using current approaches, requires extensive fractionation, separation, and enrichment. Over the years, organelle separation was achieved through the use of differential and density-gradient ultracentrifugation. However, the traditional fixed-volume process is a time-consuming and labor-intensive method, especially with large quantities of sample. Here, we present a novel tool for subcellular fractionation of biologically complex mixtures: continuous-flow ultracentrifugation of tissue homogenates to obtain both organelle separation and extensive organelle enrichment at the same time. In this study, rat liver tissues from two different age groups (3-8 wk and greater than 1 y old) were homogenized by blending. After removing nuclei, the resulting homogenates were further fractionated at the subcellular level by the use of sucrose gradient continuous-flow ultracentrifugation. Each organelle's enriched fractions were identified by Western blot analysis. To study the possible effects of aging on the endoplasmic reticulum and Golgi apparatus, we compared the organelle protein profiles of the two groups of rat liver tissues using two-dimensional gel electrophoresis, digitized imaging of two-dimensional gel electrophoresis, and mass spectrometry. Significant differences in the protein profiles of both organelles were observed between the two groups of rat tissues. The technique described here for fractionation and enrichment of organelles demonstrated a useful tool for proteomics research, including identification of low-abundance proteins and post-translational modifications.
Drahos
Kate L
KL
Alfa Wassermann Proteomic Technologies, LLC, West Caldwell, NJ 07006, USA.
Tran
Hung-Cuong
HC
Kiri
Ajay N
AN
Lan
Wenkui
W
McRorie
Donald K
DK
Horn
Marcus J
MJ
D000375:Aging / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D006056:Golgi Apparatus / Q000737:chemistry*; D006868:Hydrolysis; D008110:Liver Extracts / Q000032:analysis / Q000737:chemistry*; D008297:Male; D013058:Mass Spectrometry; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization
D008110:Liver Extracts; D011506:Proteins