Department of Chemistry, University of California, Riverside, Riverside, California 92521, United States.
Proteomics Core, Institute for Integrative Genome Biology, University of California, Riverside, Riverside, California 92521, United States.
Anal Chem. 2021 Oct 5;93(39):13398-13406. doi: 10.1021/acs.analchem.1c03481. Epub 2021 Sep 24.
DNA-protein cross-links have broad applications in mapping DNA-protein interactions and provide structural insights into macromolecular structures. However, high-resolution mapping of DNA-interacting amino acid residues with tandem mass spectrometry remains challenging due to difficulties in sample preparation and data analysis. Herein, we developed a method for identifying cross-linking amino residues in DNA-protein cross-links at single amino acid resolution. We leveraged the alkaline lability of ribonucleotides and designed ribonucleotide-containing DNA to produce structurally defined nucleic acid-peptide cross-links under our optimized ribonucleotide cleavage conditions. The structurally defined oligonucleotide-peptide heteroconjugates improved ionization, reduced the database search space, and facilitated the identification of cross-linking residues in peptides. We applied the workflow to identifying abasic (AP) site-interacting residues in human mitochondrial transcription factor A (TFAM)-DNA cross-links. With sub-nmol sample input, we obtained high-quality fragmentation spectra for nucleic acid-peptide cross-links and identified 14 cross-linked lysine residues with the home-built AP_CrosslinkFinder program. Semi-quantification based on integrated peak areas revealed that K186 of TFAM is the major cross-linking residue, consistent with K186 being the closest (to the AP modification) lysine residue in solved TFAM:DNA crystal structures. Additional cross-linking lysine residues (K69, K76, K136, K154) support the dynamic characteristics of TFAM:DNA complexes. Overall, our combined workflow using ribonucleotide as a chemically cleavable DNA modification together with optimized sample preparation and data analysis offers a simple yet powerful approach for mapping cross-linking sites in DNA-protein cross-links. The method is amendable to other chemical or photo-cross-linking systems and can be extended to complex biological samples.
DNA-蛋白质交联在绘制 DNA-蛋白质相互作用图谱和提供大分子结构的结构见解方面具有广泛的应用。然而，由于样品制备和数据分析的困难，使用串联质谱法高分辨率地绘制与 DNA 相互作用的氨基酸残基的交联仍然具有挑战性。在此，我们开发了一种方法，可在单个氨基酸分辨率下鉴定 DNA-蛋白质交联中的交联氨基酸残基。我们利用核苷酸的碱性不稳定性，并设计了含有核苷酸的 DNA，以在我们优化的核苷酸切割条件下产生结构明确的核酸-肽交联。结构明确的寡核苷酸-肽杂种提高了离子化效率，减少了数据库搜索空间，并有助于鉴定肽中的交联残基。我们将该工作流程应用于鉴定人线粒体转录因子 A（TFAM）-DNA 交联物中的无碱基（AP）位点相互作用残基。通过亚纳摩尔样品输入，我们获得了核酸-肽交联的高质量碎裂光谱，并使用我们自己开发的 AP_CrosslinkFinder 程序鉴定了 14 个交联赖氨酸残基。基于积分峰面积的半定量分析表明，TFAM 的 K186 是主要的交联残基，与在已解决的 TFAM:DNA 晶体结构中 K186 是最接近（AP 修饰）的赖氨酸残基的结果一致。其他交联赖氨酸残基（K69、K76、K136、K154）支持 TFAM:DNA 复合物的动态特性。总体而言，我们使用核苷酸作为可化学切割的 DNA 修饰物的组合工作流程，以及优化的样品制备和数据分析，为在 DNA-蛋白质交联中绘制交联位点提供了一种简单而强大的方法。该方法适用于其他化学或光交联系统，并可扩展到复杂的生物样品。
