Alternative pre-mRNA splicing has long been proposed to contribute greatly to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)-based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom peptide database trained on analysis of RNA-seq data with MAJIQ, an algorithm optimized to detect and quantify differential and unannotated splice junction usage. We matched tandem mass spectra acquired by data-dependent acquisition (DDA) against our custom RNA-seq based database, as well as SWISS-PROT and RefSeq databases to improve identification of splicing-derived proteoforms by 28% compared with use of the SWISS-PROT database alone. Altogether, we identified peptide evidence for 554 alternate proteoforms corresponding to 274 genes. Our increased depth and detection of proteins also allowed us to track changes in the transcriptome and proteome induced by T-cell stimulation, as well as fluctuations in protein subcellular localization. In sum, our data here confirm that use of generic databases in proteomic studies underestimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.

中文翻译：

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深度剖析和自定义数据库改进了对可变剪接生成的蛋白质组的检测。

长期以来，人们一直提出替代性前体 mRNA 剪接对蛋白质组的复杂性有很大贡献。然而，成熟的 mRNA 异构体在多大程度上成功地转化为蛋白质仍然存在争议。在这里，我们使用高通量 RNA 测序和基于质谱 (MS) 的蛋白质组学来更好地评估可变剪接 mRNA 的翻译。为了增加蛋白质组覆盖率并改进蛋白质定量，我们在蛋白质和肽水平上优化了细胞分级分离和样品处理步骤。此外，我们生成了一个自定义肽数据库，该数据库经过 MAJIQ 分析 RNA-seq 数据的训练，MAJIQ 是一种优化的算法，用于检测和量化差异和未注释的剪接点使用情况。我们将通过数据相关采集 (DDA) 获得的串联质谱与我们定制的基于 RNA-seq 的数据库进行匹配，以及 SWISS-PROT 和 RefSeq 数据库，与单独使用 SWISS-PROT 数据库相比，可将剪接衍生蛋白质组的识别提高 28%。总之，我们确定了对应于 274 个基因的 554 种替代蛋白质形式的肽证据。我们增加的蛋白质深度和检测也使我们能够追踪由 T 细胞刺激引起的转录组和蛋白质组的变化，以及蛋白质亚细胞定位的波动。总之，我们这里的数据证实，在蛋白质组学研究中使用通用数据库低估了被翻译成蛋白质的剪接 mRNA 异构体的数量，并提供了一个改进大规模蛋白质组学实验中异构体检测的工作流程。