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CHO cells were synchronized at metaphase by successive thymidine and nocodazole blocks. Taxol was applied to stabilize the spindle structure. Cells were lysed in a hypotonic solution to release spindles. Four spindle samples were each trypsinized and MudPIT was performed. Proteins were identified by tandem liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS), and the metaphase spindle proteome was assembled. Proteins in at least three mass spec runs appeared in the final list.