Abstract: Objective To develop a method for extracting cytoskeletons and cytoskeleton-associated proteins for proteomic
analysis. Methods A subcellular sequential proteome extraction method was exploited. The extraction procedure was optimized
and controlled according to observed cell morphology changes and one- and two-dimensional electrophoresis images. The
extraction efficiency and selectivity were evaluated by Western blotting and mass spectrometry. Results Four extracted fractions
clearly displayed distinct patterns. Western blotting detected the fraction-marker proteins FAK, intergrin-β1, histone H1 and
cytokeratin 19 only in their expected fractions. About 90% of the protein spots in the cytoskeleton fraction were identified by
mass spectrometry as cytoskeleton and/or its associated proteins. Conclusion The subcellular proteome sequential fractionation
method facilitates the detection of proteins of low abundance and shows a high reproducibility and selectivity, and thus can
serve as an ideal pre-fractionation method prior to two-dimensional electrophoresis.