Optimization of Experimental Parameters in Data-Independent Mass Spectrometry Significantly Increases Depth and Reproducibility of Results.
Optimization of Experimental Parameters in Data-Independent Mass Spectrometry Significantly Increases Depth and Reproducibility of Results.
复制标题
DOI：
10.1074/mcp.ra117.000314
复制
发表时间：
2017-12
期刊：
影响因子：
--
通讯作者：
Reiter L
中科院分区：
文献类型：
--
作者：
Bruderer R;Bernhardt OM;Gandhi T;Xuan Y;Sondermann J;Schmidt M;Gomez-Varela D;Reiter L
对大样本群体进行全面、可重复和精确的分析是定量蛋白质组学的关键目标之一。在这里，我们提出了一个实现的数据独立的采集使用其并行采集的性质，超越了串行MS 2采集的数据依赖的四极超高场轨道阱质谱仪上的采集的限制。在深度单次数据独立采集中，我们使用2个或更多肽/蛋白质鉴定并定量了人类细胞系中的6，383种蛋白质，当包括基于单个肽序列鉴定的717种蛋白质时，鉴定并定量了超过7100种蛋白质。使用2个或更多个肽/蛋白质在小鼠组织中鉴定了7739个蛋白质，并且当包括基于单个肽序列鉴定的382个蛋白质时，鉴定了8121个蛋白质。蛋白质的缺失值在0.3%至2.1%范围内，技术性三次重复测定的变异系数中位数为4.7%至6.2%。在非常复杂的混合物中，我们可以定量10，780种蛋白质和12，192种蛋白质，其中包括基于单个肽序列鉴定的1412种蛋白质。使用这个优化的DIA，我们调查了大蛋白网络之前和之后的关键时期的胡须经验诱导的突触强度在小鼠体感皮层1桶场。这项工作表明，并行质谱技术能够以高覆盖率，可重复性，精确性和可扩展性发现蛋白质组谱。
Comprehensive, reproducible and precise analysis of large sample cohorts is one of the key objectives of quantitative proteomics. Here, we present an implementation of data-independent acquisition using its parallel acquisition nature that surpasses the limitation of serial MS2 acquisition of data-dependent acquisition on a quadrupole ultra-high field Orbitrap mass spectrometer. In deep single shot data-independent acquisition, we identified and quantified 6,383 proteins in human cell lines using 2-or-more peptides/protein and over 7100 proteins when including the 717 proteins that were identified on the basis of a single peptide sequence. 7739 proteins were identified in mouse tissues using 2-or-more peptides/protein and 8121 when including the 382 proteins that were identified based on a single peptide sequence. Missing values for proteins were within 0.3 to 2.1% and median coefficients of variation of 4.7 to 6.2% among technical triplicates. In very complex mixtures, we could quantify 10,780 proteins and 12,192 proteins when including the 1412 proteins that were identified based on a single peptide sequence. Using this optimized DIA, we investigated large-protein networks before and after the critical period for whisker experience-induced synaptic strength in the murine somatosensory cortex 1-barrel field. This work shows that parallel mass spectrometry enables proteome profiling for discovery with high coverage, reproducibility, precision and scalability.