Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer
Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer
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DOI：
10.1021/acs.jproteome.6b00698
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发表时间：
2017-02-01
影响因子：
4.4
通讯作者：
Kelleher, Neil L.
中科院分区：
文献类型：
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作者：
Fornelli, Luca;Durbin, Kenneth R.;Kelleher, Neil L.
在过去的十年中，高分辨率质谱的发展使得能够从复杂蛋白质组中高通量分析完整蛋白质，从而鉴定数千种蛋白质型。先前关于自上而下蛋白质组学（TDP）的几个报告依赖于混合离子阱傅立叶变换质谱仪结合数据依赖的采集策略。为了进一步减少TDP的实践，我们使用四极杆Orbitrap仪器与软件相结合，用于蛋白质型依赖性数据采集，以1%的错误发现率从人成纤维细胞中识别和表征近2000种蛋白质型。通过将3 m/z分离窗口与短瞬变相结合以提高>30 kDa的蛋白质型的特异性和信噪比，我们证明了通过捕获30-60 kDa范围内的439种蛋白质型来提高蛋白质组覆盖率。三种不同的数据采集策略进行了比较，并导致在重复数据依赖性实验中未观察到的许多proteoforms的鉴定。值得注意的是，数据集用更新的度量和工具报告，包括新的查看器和用于包含高度表征的蛋白质形式的永久蛋白质形式记录标识符的分配（即，C-分数>40的那些）在由自上而下蛋白质组学联盟（Consortium for Top-Down Proteomics）策划的储存库中。
Over the past decade, developments in high resolution mass spectrometry have enabled the high throughput analysis of intact proteins from complex proteomes, leading to the identification of thousands of proteoforms. Several previous reports on top-down proteomics (TDP) relied on hybrid ion trap Fourier transform mass spectrometers combined with data-dependent acquisition strategies. To further reduce TDP to practice, we use a quadrupole-Orbitrap instrument coupled with software for proteoform-dependent data acquisition to identify and characterize nearly 2000 proteoforms at a 1% false discovery rate from human fibroblasts. By combining a 3 m/z isolation window with short transients to improve specificity and signal-to-noise for proteoforms >30 kDa, we demonstrate improving proteome coverage by capturing 439 proteoforms in the 30-60 kDa range. Three different data acquisition strategies were compared and resulted in the identification of many proteoforms not observed in replicate data-dependent experiments. Notably, the data set is reported with updated metrics and tools including a new viewer and assignment of permanent proteoform record identifiers for inclusion of highly characterized proteoforms (i.e., those with C-scores >40) in a repository curated by the Consortium for Top-Down Proteomics.