A Fast Workflow for Identification and Quantification of Proteomes
A Fast Workflow for Identification and Quantification of Proteomes
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用于蛋白质组识别和定量的快速工作流程。
DOI：
10.1074/mcp.o112.025023
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发表时间：
2013-08-01
影响因子：
7
通讯作者：
Qin, Jun
中科院分区：
文献类型：
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作者：
Ding, Chen;Jiang, Jing;Qin, Jun
目前深入的蛋白质组学利用长色谱梯度来获得更多的肽用于蛋白质鉴定，导致在3天的质谱运行时间内覆盖多达8000个哺乳动物基因产物。在这里，我们报告了使用双反相高效液相色谱-质谱（HPLC-MS）的快速测序（Fast-seq）工作流程，其具有短梯度，以在0.5天内实现相同的蛋白质组覆盖率。我们将此工作流程调整为定量版本（快速定量，Fast-quan），该版本与大规模蛋白质定量兼容。我们对两个相同的样本进行了Fast-quan工作流程，这使我们能够系统地评估影响工作流程灵敏度和准确性的不同参数。利用显著性检验的统计量，我们揭示了大量错误定量差异蛋白的存在，并估计了错误定量率与无标记定量参数的相关性。我们优化了参数的设置，可以大大减少错误定量的差异蛋白质的比率，并进一步将其应用于真实的生物过程。随着效率和通量的提高，我们预计Fast-seq/Fast-quan工作流程，允许在1天内对两个蛋白质组进行配对比较，可以使MS可用于大众，并以积极的方式影响生物医学研究。
The current in-depth proteomics makes use of long chromatography gradient to get access to more peptides for protein identification, resulting in covering of as many as 8000 mammalian gene products in 3 days of mass spectrometer running time. Here we report a fast sequencing (Fast-seq) workflow of the use of dual reverse phase high performance liquid chromatography - mass spectrometry (HPLC-MS) with a short gradient to achieve the same proteome coverage in 0.5 day. We adapted this workflow to a quantitative version (Fast quantification, Fast-quan) that was compatible to large-scale protein quantification. We subjected two identical samples to the Fast-quan workflow, which allowed us to systematically evaluate different parameters that impact the sensitivity and accuracy of the workflow. Using the statistics of significant test, we unraveled the existence of substantial falsely quantified differential proteins and estimated correlation of false quantification rate and parameters that are applied in label-free quantification. We optimized the setting of parameters that may substantially minimize the rate of falsely quantified differential proteins, and further applied them on a real biological process. With improved efficiency and throughput, we expect that the Fast-seq/Fast-quan workflow, allowing pair wise comparison of two proteomes in 1 day may make MS available to the masses and impact biomedical research in a positive way.