Proteins form adducts with nucleic acids in a variety of contexts, and these adducts may be cytotoxic if not repaired. Here we apply a proteomic approach to identification of proteins adducted to DNA or RNA in normally proliferating cells. This approach combines RADAR fractionation of proteins covalently bound to nucleic acids with quantitative mass spectrometry (MS). We demonstrate that "RADAR-MS" can quantify induction of TOP1- or TOP2-DNA adducts in cells treated with topotecan or etoposide, respectively, and also identify intermediates in physiological adduct repair. We validate RADAR-MS for discovery of previously unknown adducts by determining the repertoires of adducted proteins in two different normally proliferating human cell lines, CCRF-CEM T cells and GM639 fibroblasts. These repertoires are significantly similar with one another and exhibit robust correlations in their quantitative profiles (Spearman r = 0.52). A very similar repertoire is identified by the classical approach of CsCl buoyant density gradient centrifugation. We find that in normally proliferating human cells, the repertoire of adducted proteins - the "adductome" - is comprised of a limited number of proteins belonging to specific functional groups, and that it is greatly enriched for histones, HMG proteins and proteins involved in RNA splicing. Treatment with low concentrations of formaldehyde caused little change in the composition of the repertoire of adducted proteins, suggesting that reactive aldehydes generated by ongoing metabolic processes may contribute to protein adduction in normally proliferating cells. The identification of an endogenous adductome highlights the importance of adduct repair in maintaining genomic structure and the potential for deficiencies in adduct repair to contribute to cancer.

中文翻译：

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“加合物”：人类细胞中有限的加合蛋白库。

蛋白质在各种情况下与核酸形成加合物，如果不修复，这些加合物可能具有细胞毒性。在这里，我们应用蛋白质组学方法来鉴定在正常增殖细胞中与 DNA 或 RNA 加合的蛋白质。这种方法将与核酸共价结合的蛋白质的雷达分离与定量质谱 (MS) 相结合。我们证明了“RADAR-MS”可以分别量化用拓扑替康或依托泊苷处理的细胞中 TOP1-或 TOP2-DNA 加合物的诱导，并鉴定生理加合物修复中的中间体。我们通过确定两种不同的正常增殖的人类细胞系 CCRF-CEM T 细胞和 GM639 成纤维细胞中的加合物蛋白库来验证 RADAR-MS 发现以前未知的加合物。这些曲目彼此显着相似，并且在它们的定量概况中表现出强大的相关性（Spearman r = 0.52）。通过 CsCl 浮力密度梯度离心的经典方法确定了一个非常相似的曲目。我们发现，在正常增殖的人类细胞中，加合物的所有蛋白质 - “加合物” - 由属于特定功能组的有限数量的蛋白质组成，并且它富含组蛋白、HMG 蛋白和参与 RNA 的蛋白质拼接。用低浓度甲醛处理导致加合蛋白质的组成几乎没有变化，这表明正在进行的代谢过程产生的反应性醛可能有助于正常增殖细胞中的蛋白质加合。