Isolation of mitotic chromosomes from vertebrate cells and characterisation of their proteome by mass spectrometry

Chromosomes consist of enormously long DNA molecules plus the proteins that package and regulate the transcription and replication of this DNA. In order to understand both the composition of the bulk chromatin that packages the DNA and the specialized structures that direct its segregation (e.g., centromeres and kinetochores), one requirement is to have a list of the component proteins of mitotic chromosomes. Identification and quantitation of these proteins and their modifications require the ability to isolate chromosomes and analyze their proteome by mass spectrometry. Here, we describe a step-by-step protocol to isolate mitotic chromosomes from vertebrate cells. The chromosome proteins may be labeled in vivo with heavy stable isotope for quantitative proteomics. We then go through the proteomics workflow from preparation of samples to their analysis in the mass spectrometer. Finally, we describe some of the software used in processing of output data for statistical and bioinformatic analysis.