Evaluation of Sample Disruption Techniques for High Throughput Extraction of Subcellular Organelles

Abstract

Modern mass spectrometers coupled with high resolution separation strategies have enabled the quantification of thousands of proteins from minute samples. However, the large dynamic range in protein abundances, the presence of sequence variants and post‐translational modifications often require that subcellular fractionation strategies be performed in order to achieve the necessary sequence coverage and signal intensity to elucidate statistically significant changes in protein expression. Subcellular fractionation enables the organelle constituents to be separated prior to downstream enzyme digestion and proteomic analysis to increase the detection of proteins of lower abundance thus increasing proteome coverage. Subcellular fractionation encompasses at least two steps; sample disruption (homogenization) and a fractionation procedure. While many organelle fractionation procedures have been described, including density centrifugation, there is no single technique that has been demonstrated to produce an ideal homogenate containing a suspension of separated intact organelles. Here we evaluate the use of three common homogenizer technologies, dounce, rotor‐stator and bead milling for the disruption of tissues for the purpose of isolating commonly analyzed organelles, including nuclei, plasma membrane, mitochondria, endoplasmic reticulum and cytosol. Mouse brain, liver and heart tissues are processed using increasing degrees of disruption forces then homogenates were separated by density centrifugation and analyzed by microscopy, western blotting and bottom up proteomics to evaluate the degree of organelle enrichment.