Wellcome Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, University of Edinburgh. Edinburgh EH9 3BF, Scotland, UK.
Nucleic Acids Res. 2024 Feb 28;52(4):e21. doi: 10.1093/nar/gkad1249.
The RNA-interacting proteome is commonly characterized by UV-crosslinking followed by RNA purification, with protein recovery quantified using SILAC labeling followed by data-dependent acquisition (DDA) of proteomic data. However, the low efficiency of UV-crosslinking, combined with limited sensitivity of the DDA approach often restricts detection to relatively abundant proteins, necessitating multiple mass spec injections of fractionated peptides for each biological sample. Here we report an application of data-independent acquisition (DIA) with SILAC in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment. This gave 15% greater protein detection and lower inter-replicate variation relative to the same biological materials analyzed using DDA, while allowing single-shot analysis of the sample. As proof of concept, we determined the effects of arsenite treatment on the RNA-bound proteome of HEK293T cells. The DIA dataset yielded similar GO term enrichment for RNA-binding proteins involved in cellular stress responses to the DDA dataset while detecting extra proteins unseen by DDA. Overall, the DIA SILAC approach improved detection of proteins over conventional DDA SILAC for generating RNA-interactome datasets, at a lower cost due to reduced machine time. Analyses are described for TRAPP data, but the approach is suitable for proteomic analyses following essentially any RNA-binding protein enrichment technique.
RNA 相互作用蛋白质组学通常通过紫外线交联进行特征描述，然后进行 RNA 纯化，使用 SILAC 标记定量回收蛋白质，然后对蛋白质组学数据进行依赖于数据的获取（DDA）。然而，紫外线交联的效率低，再加上 DDA 方法的灵敏度有限，通常限制了对相对丰富蛋白质的检测，需要对每个生物样品进行多次分馏肽的质谱注射。在这里，我们报告了在总 RNA 相关蛋白纯化（TRAPP）紫外线交联实验中使用 SILAC 的无依赖数据获取（DIA）的应用。与使用 DDA 分析相同生物材料相比，DIA 增加了 15%的蛋白质检测，并且降低了重复间的差异，同时允许对样品进行单次分析。作为概念验证，我们确定了亚砷酸盐处理对 HEK293T 细胞中 RNA 结合蛋白组的影响。DIA 数据集在检测到 DDA 无法检测到的额外蛋白质的同时，产生了与 DDA 数据集相似的 GO 术语富集，用于参与细胞应激反应的 RNA 结合蛋白。总体而言，与传统的 DDA SILAC 相比，DIA SILAC 方法在生成 RNA 相互作用数据集方面提高了蛋白质的检测，由于机器时间减少，成本降低。对 TRAPP 数据进行了分析，但该方法适用于本质上任何 RNA 结合蛋白富集技术之后的蛋白质组学分析。
