Chromatin-Associated Proteins Revealed by SILAC-Proteomic Analysis Exhibit a High Likelihood of Requirement for Growth Fitness under DNA Damage Stress

Abstract EN

Chromatin-associated nonhistone proteins (CHRAPs) are readily collected from the DNaseI digested crude chromatin preparation.

In this study, we show that the absolute abundance-based label-free quantitative proteomic analysis fail to identify potential CHRAPs from the CHRAP-prep.

This is because that the most-highly abundant cytoplasmic proteins such as ribosomal proteins are not effectively depleted in the CHRAP-prep.

Ribosomal proteins remain the top-ranked abundant proteins in the CHRAP-prep.

On the other hand, we show that relative abundance-based SILAC-mediated quantitative proteomic analysis is capable of discovering the potential CHRAPs in the CHRAP-prep when compared to the whole-cell-extract.

Ribosomal proteins are depleted from the top SILAC ratio-ranked proteins.

In contrast, nucleus-localized proteins or potential CHRAPs are enriched in the top SILAC-ranked proteins.

Consistent with this, gene-ontology analysis indicates that CHRAP-associated functions such as transcription, regulation of chromatin structures, and DNA replication and repair are significantly overrepresented in the top SILAC-ranked proteins.

Some of the novel CHRAPs are confirmed using the traditional method.

Notably, phenotypic assessment reveals that the top SILAC-ranked proteins exhibit the high likelihood of requirement for growth fitness under DNA damage stress.

Taken together, our results indicate that the SILAC-mediated proteomic approach is capable of determining CHRAPs without prior knowledge.