Revealing Novel Telomere Proteins Using in Vivo Cross-linking, Tandem Affinity Purification, and Label-free Quantitative LC-FTICR-MS
Revealing Novel Telomere Proteins Using in Vivo Cross-linking, Tandem Affinity Purification, and Label-free Quantitative LC-FTICR-MS
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DOI：
10.1074/mcp.m900490-mcp200
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发表时间：
2010-06-01
影响因子：
7
通讯作者：
Stewart, Sheila A.
中科院分区：
文献类型：
--
作者：
Nittis, Thalia;Guittat, Lionel;Stewart, Sheila A.
端粒是 DNA 蛋白质结构，可保护染色体末端免受 DNA 修复机制的影响。当端粒完整性受到损害时，基因组就会不稳定。相当多的努力集中在端粒结合蛋白的鉴定和其功能的阐明上。迄今为止，蛋白质鉴定依赖于经典的免疫沉淀和质谱方法，主要是在有利于分离具有强或长期相互作用的蛋白质的条件下，这些蛋白质的存在量足以通过 SDS-PAGE 可视化。为了便于鉴定低丰度和瞬时相关的端粒结合蛋白，我们开发了一种新方法，结合了体内蛋白质-蛋白质交联、串联亲和纯化和严格的顺序内切蛋白酶消化。通过无标记比较纳米 LC-FTICR-MS 鉴定肽。在这里，我们表达了表位标记的端粒结合蛋白，并利用改良的染色质免疫沉淀方法来交联相关蛋白。所得免疫沉淀剂含有端粒 DNA，证实该方法捕获了真正的端粒结合复合物。为了鉴定免疫捕获复合物中存在的蛋白质，通过连续的内切蛋白酶处理对样品进行还原、烷基化和消化。使用微米级多孔石墨固定相纯化所得肽，并使用纳米 LC-FTICR-MS 进行分析。通过对不同样品的 FTICR 质谱进行无标记定量分析和离子阱串联质谱分析，然后进行数据库搜索，鉴定了表达 HA-FLAG-TIN2 的细胞中富集的蛋白质。我们鉴定了构成端粒庇护蛋白复合物的所有蛋白质，从而验证了该方法的稳健性。我们还鉴定了 62 种新型端粒结合蛋白。这些结果表明，DNA 结合的蛋白质复合物，包括那些以低摩尔比存在的蛋白质复合物，可以通过这种方法进行鉴定。这种方法的成功将使我们对端粒维持有更全面的了解并具有广泛的适用性。分子与细胞蛋白质组学 9：1144-1156，2010。
Telomeres are DNA-protein structures that protect chromosome ends from the actions of the DNA repair machinery. When telomeric integrity is compromised, genomic instability ensues. Considerable effort has focused on identification of telomere-binding proteins and elucidation of their functions. To date, protein identification has relied on classical immunoprecipitation and mass spectrometric approaches, primarily under conditions that favor isolation of proteins with strong or long lived interactions that are present at sufficient quantities to visualize by SDS-PAGE. To facilitate identification of low abundance and transiently associated telomere-binding proteins, we developed a novel approach that combines in vivo protein-protein cross-linking, tandem affinity purification, and stringent sequential endoprotease digestion. Peptides were identified by label-free comparative nano-LC-FTICR-MS. Here, we expressed an epitope-tagged telomere-binding protein and utilized a modified chromatin immunoprecipitation approach to cross-link associated proteins. The resulting immunoprecipitant contained telomeric DNA, establishing that this approach captures bona fide telomere binding complexes. To identify proteins present in the immunocaptured complexes, samples were reduced, alkylated, and digested with sequential endoprotease treatment. The resulting peptides were purified using a microscale porous graphite stationary phase and analyzed using nano-LC-FTICR-MS. Proteins enriched in cells expressing HA-FLAG-TIN2 were identified by label-free quantitative analysis of the FTICR mass spectra from different samples and ion trap tandem mass spectrometry followed by database searching. We identified all of the proteins that constitute the telomeric shelterin complex, thus validating the robustness of this approach. We also identified 62 novel telomere-binding proteins. These results demonstrate that DNA-bound protein complexes, including those present at low molar ratios, can be identified by this approach. The success of this approach will allow us to create a more complete understanding of telomere maintenance and have broad applicability. Molecular & Cellular Proteomics 9:1144-1156, 2010.