Surpassing 10 000 identified and quantified proteins in a single run by optimizing current LC-MS instrumentation and data analysis strategy
Surpassing 10 000 identified and quantified proteins in a single run by optimizing current LC-MS instrumentation and data analysis strategy
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DOI：
10.1039/c9mo00082h
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发表时间：
2019-10-01
期刊：
影响因子：
2.9
通讯作者：
Reiter, Lukas
中科院分区：
文献类型：
--
作者：
Muntel, Jan;Gandhi, Tejas;Reiter, Lukas
全面的蛋白质组定量对于更好地理解疾病的潜在机制至关重要。液相色谱质谱法（LC-MS）由于其强大的功能和多功能性，已成为全面蛋白质组定量的首选方法。尽管近年来已经取得了很大的进展，但由于蛋白质表达的高动态范围，复杂样品的全蛋白质组覆盖仍然具有挑战性。此外，当研究疾病调节蛋白时，生物标志物或潜在的药物靶标通常是低丰度的，例如激酶和转录因子。在这里，我们表明，随着色谱和数据分析的改进，单次蛋白质组覆盖率可以超过10 000个蛋白质在人体组织中。在睾丸癌研究中，我们使用数据独立采集（DIA）定量了11200种蛋白质。该深度是以1%的错误发现率实现的，这是使用两个物种测试进行实验验证的。我们引入了混合图书馆的概念，它结合了直接搜索DIA数据的优势，以及使用大型项目特定的或已发布的DDA数据集。使用混合文库可以实现非常深的蛋白质组覆盖，而无需创建项目特定文库的额外负担。在睾丸癌组中，我们发现大部分蛋白质的表达发生了改变（总计：3351; 1453在癌症中增加）。其中许多蛋白质可能与癌症的标志有关。例如，补体系统下调，这有助于逃避免疫应答，染色体复制上调，表明细胞周期失调。
Comprehensive proteome quantification is crucial for a better understanding of underlying mechanisms of diseases. Liquid chromatography mass spectrometry (LC-MS) has become the method of choice for comprehensive proteome quantification due to its power and versatility. Even though great advances have been made in recent years, full proteome coverage for complex samples remains challenging due to the high dynamic range of protein expression. Additionally, when studying disease regulatory proteins, biomarkers or potential drug targets are often low abundant, such as for instance kinases and transcription factors. Here, we show that with improvements in chromatography and data analysis the single shot proteome coverage can go beyond 10 000 proteins in human tissue. In a testis cancer study, we quantified 11 200 proteins using data independent acquisition (DIA). This depth was achieved with a false discovery rate of 1% which was experimentally validated using a two species test. We introduce the concept of hybrid libraries which combines the strength of direct searching of DIA data as well as the use of large project-specific or published DDA data sets. Remarkably deep proteome coverage is possible using hybrid libraries without the additional burden of creating a project-specific library. Within the testis cancer set, we found a large proportion of proteins in an altered expression (in total: 3351; 1453 increased in cancer). Many of these proteins could be linked to the hallmarks of cancer. For example, the complement system was downregulated which helps to evade the immune response and chromosomal replication was upregulated indicating a dysregulated cell cycle.