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. 1. Background Chromatin is a complex of DNA and proteins, in which the histones H2A, H2B, H3, and H4 are the major protein constituents [1, 2]. Chromatin remodeling through posttranslational modification of histones plays an important role in modulation of DNA-protein interaction and thus regulates various biological processes such as replication, DNA damage repair, and transcription [3]. Hence, identification of the chromatin associated nonhistone proteins (CHRAPs) would permit understanding the molecular mechanisms for chromatin remodeling and regulation of various biological processes. Fission yeast is a useful model for analysis of RNA interference (RNAi) directed heterochromatin formation [4, 5]. Many CHRAPs have been identified by using the high-throughput proteomic analysis of protein complexes purified through the chromatin immunoprecipitation (ChIP) coupled with the tandem affinity protein purification (TAP) tagging method in which the known CHRAP is used as bait [6–12]. However, it is limited to the identification of the CHRAPs that are associated with the complexes
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