Sucrose gradient chromatin enrichment for quantitative proteomics analysis in budding yeast

Area of Science:

• Molecular Biology
• Proteomics
• Cellular Biology

Background:

• The chromatin-bound proteome (chromatome) plays a crucial role in regulating DNA-related processes.
• Understanding changes in chromatome composition is vital for deciphering cellular responses to DNA damage.
• Existing methods may have limitations in quantifying these dynamic changes.

Purpose of the Study:

• To develop and optimize a robust protocol for quantifying relative abundance changes in the chromatin-bound proteome.
• To apply this protocol to investigate alterations in chromatin composition following DNA damage.
• To provide a detailed methodology for reproducible proteomic analysis of the chromatome.

Main Methods:

• Development of a stringent chromatin fractionation protocol.
• Application of tandem mass tag (TMT) multiplexing-based tandem mass spectrometry for quantitative proteomic analysis.
• Systematic evaluation of the fractionation and mass spectrometry procedures.

Main Results:

• The optimized protocol enables precise quantification of changes in the chromatin proteome.
• Application to yeast cells exposed to DNA-damaging drugs revealed significant alterations in chromatin composition.
• The study successfully characterized the impact of the DNA damage response on the chromatome.

Conclusions:

• The described fractionation protocol is effective for studying DNA damage-induced changes in the chromatin proteome.
• This methodology provides valuable insights into the dynamic nature of chromatin composition under cellular stress.
• The protocol serves as a foundation for future research into DNA repair mechanisms and chromatin regulation.