Improved identification of enriched peptide RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry.
Improved identification of enriched peptide RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry.
复制标题
DOI：
10.1093/nar/gkm540
复制
发表时间：
2007
影响因子：
14.9
通讯作者：
Urlaub H
中科院分区：
文献类型：
--
作者：
Kühn-Hölsken E;Dybkov O;Sander B;Lührmann R;Urlaub H
直接UV交联结合质谱（MS）是鉴定天然核糖核蛋白颗粒（RNP）如剪接体中迄今未表征的蛋白质-RNA接触位点的有力工具。交联后接触部位的识别受到以下因素的限制：（i）交联产率相对较低，以及（ii）可用于交联研究的起始材料的量。因此，这种分析中最关键的步骤是在MS分析之前从过量的非交联材料中广泛纯化交联的肽-RNA杂缀合物。在这里，我们描述了一种策略，结合小规模的反相液相色谱法（RP-HPLC）的UV照射和水解的RNP，固定化金属离子亲和色谱法（IMAC），以丰富交联的物种和他们的分析基质辅助激光解吸/电离（MALDI）MS（/MS）。在无法进行MS/MS分析的情况下，用碱性磷酸酶处理富集的馏分可明确鉴定交联物质。我们证明了这种策略的可行性，通过MS分析富集肽-RNA交联从UV照射重建[15.5K-61 K-U4 atac snRNA] snRNP和天然U1 snRNP。将我们的方法应用于U2 snRNP的部分复合物，使我们能够识别U2 snRNP特异性蛋白p14/SF 3b 14 a和U2 snRNA的分支位点相互作用区（BSiR）之间的接触位点。
Direct UV cross-linking combined with mass spectrometry (MS) is a powerful tool to identify hitherto non-characterized protein–RNA contact sites in native ribonucleoprotein particles (RNPs) such as the spliceosome. Identification of contact sites after cross-linking is restricted by: (i) the relatively low cross-linking yield and (ii) the amount of starting material available for cross-linking studies. Therefore, the most critical step in such analyses is the extensive purification of the cross-linked peptide–RNA heteroconjugates from the excess of non-crosslinked material before MS analysis. Here, we describe a strategy that combines small-scale reversed-phase liquid chromatography (RP-HPLC) of UV-irradiated and hydrolyzed RNPs, immobilized metal-ion affinity chromatography (IMAC) to enrich cross-linked species and their analysis by matrix-assisted laser desorption/ionisation (MALDI) MS(/MS). In cases where no MS/MS analysis can be performed, treatment of the enriched fractions with alkaline phosphatase leads to unambiguous identification of the cross-linked species. We demonstrate the feasibility of this strategy by MS analysis of enriched peptide–RNA cross-links from UV-irradiated reconstituted [15.5K-61K-U4atac snRNA] snRNPs and native U1 snRNPs. Applying our approach to a partial complex of U2 snRNP allowed us to identify the contact site between the U2 snRNP-specific protein p14/SF3b14a and the branch-site interacting region (BSiR) of U2 snRNA.
登录
查看更多内容
影响因子：
5.3
作者：
Deckert, Jochen;Hartmuth, Maus;Luehrmann, Reinhard
通讯作者：
Luehrmann, Reinhard
影响因子：
14.8
作者：
Hernandez, Helena;Robinson, Carol V.
通讯作者：
Robinson, Carol V.
影响因子：
5.3
作者：
Gozani, O;Potashkin, J;Reed, R
通讯作者：
Reed, R
影响因子：
2.9
作者：
Li, SH;Dass, C
通讯作者：
Dass, C
影响因子：
2
作者：
Janek, K;Wenschuh, H;Krause, E
通讯作者：
Krause, E