Sucrose gradient chromatin enrichment for quantitative proteomics analysis in budding yeast

Here, we describe a fractionation protocol optimized to quantify changes in relative abundance of the chromatin-bound proteome (chromatome) by tandem mass tag multiplexing-based tandem mass spectrometry. It has been applied to yeast cells before and after exposure to DNA-damaging drugs to characterize changes in chromatin composition induced by the DNA damage response. We detail steps for stringent chromatin fractionation, sample preparation for mass spectrometry, and its evaluation.For complete details on the use and execution of this protocol, please refer to Challa et al. (2021).

Automated summary

This study presents a fractionation protocol optimized for quantifying changes in the chromatin-bound proteome using tandem mass tag multiplexing-based tandem mass spectrometry. The protocol has been successfully applied to yeast cells to characterize changes induced by exposure to DNA-damaging drugs. Detailed steps for stringent chromatin fractionation, sample preparation for mass spectrometry, and evaluation are provided in the protocol.