Human Proteomic Variation Revealed by Combining RNA-Seq Proteogenomics and Global Post-Translational Modification (G-PTM) Search Strategy.
Human Proteomic Variation Revealed by Combining RNA-Seq Proteogenomics and Global Post-Translational Modification (G-PTM) Search Strategy.
复制标题
DOI：
10.1021/acs.jproteome.5b00817
复制
发表时间：
2016-03-04
影响因子：
4.4
通讯作者：
Smith LM
中科院分区：
文献类型：
--
作者：
Cesnik AJ;Shortreed MR;Sheynkman GM;Frey BL;Smith LM
由于标准蛋白质数据库中不存在变异肽和翻译后修饰 (PTM)，基于质谱的蛋白质组分析低估了蛋白质组变异。每个个体都携带数以千计的错义突变，这些突变会导致单个氨基酸变异，但这些突变却被遗漏了，因为它们在通用蛋白质组搜索数据库中不存在。多种类型的蛋白质 PTM 在生物过程中发挥着重要作用，但由于变量修饰搜索中错误发现率的增加而仍未被发现。我们用两个最近开发的软件工具解决了自下而上蛋白质组学的这两个基本缺点。第一个由 Galaxy 中的工作流程组成，该工作流程挖掘 RNA 测序数据以生成包含变体肽和选择性剪接事件产物的样本特定数据库。第二个工具采用了一种新策略，该策略改变了变量修改方法，仅考虑特定位置处的策划 PTM，从而避免了传统上导致高错误发现率的组合爆炸。使用基于 RNA 测序的数据库和这种全局翻译后修饰 (G-PTM) 搜索策略，揭示了 10 种人类细胞系中的每一种中数百种单氨基酸变异肽、数十种新型剪接肽和数百种翻译后修饰肽。
Mass-spectrometry-based proteomic analysis underestimates proteomic variation due to the absence of variant peptides and posttranslational modifications (PTMs) from standard protein databases. Each individual carries thousands of missense mutations that lead to single amino acid variants, but these are missed because they are absent from generic proteomic search databases. Myriad types of protein PTMs play essential roles in biological processes but remain undetected because of increased false discovery rates in variable modification searches. We address these two fundamental shortcomings of bottom-up proteomics with two recently developed software tools. The first consists of workflows in Galaxy that mine RNA sequencing data to generate sample-specific databases containing variant peptides and products of alternative splicing events. The second tool applies a new strategy that alters the variable modification approach to consider only curated PTMs at specific positions, thereby avoiding the combinatorial explosion that traditionally leads to high false discovery rates. Using RNA-sequencing-derived databases with this Global Post-Translational Modification (G-PTM) search strategy revealed hundreds of single amino acid variant peptides, tens of novel splice junction peptides, and several hundred posttranslationally modified peptides in each of ten human cell lines.