Assessing the Relationship Between Mass Window Width and Retention Time Scheduling on Protein Coverage for Data-Independent Acquisition
Assessing the Relationship Between Mass Window Width and Retention Time Scheduling on Protein Coverage for Data-Independent Acquisition
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DOI：
10.1007/s13361-019-02243-1
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发表时间：
2019-08-01
影响因子：
3.2
通讯作者：
Liu, Yansheng
中科院分区：
文献类型：
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作者：
Li, Wenxue;Chi, Hao;Liu, Yansheng
由于质谱仪的技术进步，特别是增加的扫描速度和更高的MS/MS分辨率，数据独立采集质谱（DIA-MS）的使用变得更加流行，这使得蛋白质组学鉴定和定量具有高重现性。目前的DIA-MS方法通常覆盖较宽的质量范围，目的是靶向和鉴定尽可能多的肽和蛋白质，因此经常生成高度复杂的MS/MS谱。在本报告中，我们评估了使用小窗口的性能和好处，例如，5-跨肽洗脱时间的m/z宽度。我们进一步设计了一种名为RTwinDIA的新DIA方法，该方法在不同的保留时间块中安排小的分离窗口，利用了较大的肽通常在反相色谱中稍后洗脱的事实。我们通过使用鸟枪数据库搜索工具（如MaxQuant和pFind）以及Spectronaut与外部人类蛋白质综合光谱库评估了直接蛋白质组学鉴定。我们的结论是，像pFind这样的算法有潜力直接分析用小窗口获得的DIA数据，并且如果优先将仪器时间和DIA周期时间花费在小窗口上而不是用大窗口覆盖广泛的质量范围上，将提高新生物样品的直接蛋白质组覆盖率并提高定量精度。这些结果进一步为DDA和DIA在更快的MS分析仪上的未来融合提供了前景。
Due to the technical advances of mass spectrometers, particularly increased scanning speed and higher MS/MS resolution, the use of data-independent acquisition mass spectrometry (DIA-MS) became more popular, which enables high reproducibility in both proteomic identification and quantification. The current DIA-MS methods normally cover a wide mass range, with the aim to target and identify as many peptides and proteins as possible and therefore frequently generate MS/MS spectra of high complexity. In this report, we assessed the performance and benefits of using small windows with, e.g., 5-m/z width across the peptide elution time. We further devised a new DIA method named RTwinDIA that schedules the small isolation windows in different retention time blocks, taking advantage of the fact that larger peptides are normally eluting later in reversed phase chromatography. We assessed the direct proteomic identification by using shotgun database searching tools such as MaxQuant and pFind, and also Spectronaut with an external comprehensive spectral library of human proteins. We conclude that algorithms like pFind have potential in directly analyzing DIA data acquired with small windows, and that the instrumental time and DIA cycle time, if prioritized to be spent on small windows rather than on covering a broad mass range by large windows, will improve the direct proteome coverage for new biological samples and increase the quantitative precision. These results further provide perspectives for the future convergence between DDA and DIA on faster MS analyzers.