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首页> 外文期刊>Journal of mass spectrometry: JMS >Systematic evaluation of data-independent acquisition for sensitive and reproducible proteomics-a prototype design for a single injection assay
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Systematic evaluation of data-independent acquisition for sensitive and reproducible proteomics-a prototype design for a single injection assay

机译：系统地评估敏感和可重现的蛋白质组学的独立于数据的采集-单次进样的原型设计

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Data-independent acquisition (DIA)-based proteomics has become increasingly complicated in recent years because of the vast number of workflows described, coupled with a lack of studies indicating a rational framework for selecting effective settings to use. To address this issue and provide a resource for the proteomics community, we compared 12 DIA methods that assay tryptic peptides using various mass-isolation windows. Our findings indicate that the most sensitive single injection LC-DIA method uses 6m/z isolation windows to analyze the densely populated tryptic peptide range from 450 to 730m/z, which allowed quantification of 4465 Escherichiacoli peptides. In contrast, using the sequential windowed acquisition of all theoretical fragment-ions (SWATH) approach with 26m/z isolation windows across the entire 400-1200m/z range, allowed quantification of only 3309 peptides. This reduced sensitivity with 26m/z windows is caused by an increase in co-eluting compounds with similar precursor values detected in the same tandem MS spectra, which lowers the signal-to-noise of peptide fragment-ion chromatograms and reduces the amount of low abundance peptides that can be quantified from 410 to 920m/z. Above 920m/z, more peptides were quantified with 26m/z windows because of substantial peptide C-13 isotope distributions that parse peptide ions into separate isolation windows. Because reproducible quantification has been a long-standing aim of quantitative proteomics, and is a so-called trait of DIA, we sought to determine whether precursor-level chromatograms used in some methods rather than their fragment-level counterparts have similar precision. Our data show that extracted fragment-ion chromatograms are the reason DIA provides superior reproducibility. Copyright (c) 2015 John Wiley & Sons, Ltd.
机译：近年来，由于所描述的工作流程众多，加上基于数据独立获取（DIA）的蛋白质组学研究变得越来越复杂，而且缺乏研究表明需要合理的框架来选择要使用的有效设置。为了解决此问题并为蛋白质组学社区提供资源，我们比较了使用各种质量隔离窗口测定胰蛋白酶肽的12种DIA方法。我们的发现表明，最灵敏的单次进样LC-DIA方法使用6m / z的隔离窗口来分析450至730m / z的密集胰蛋白酶肽段，从而可以定量4465种大肠杆菌。相反，使用在所有400-1200m / z范围内具有26m / z隔离窗的所有理论碎片离子的顺序窗口采集（SWATH）方法，仅可定量3309个肽段。在相同的串联MS质谱图中检测到的具有相似前体值的共洗脱化合物增加，导致26m / z窗口的灵敏度降低，这降低了肽片段离子色谱图的信噪比并减少了可以从410到920m / z定量的丰富多肽。高于920m / z时，由于26 C / z窗口定量了更多的肽，这是因为大量的C-13同位素分布将肽离子解析为单独的隔离窗口。由于可重现的定量化一直是定量蛋白质组学的长期目标，并且是DIA的所谓特征，因此我们试图确定某些方法中使用的前体级色谱图而不是片段级对应物是否具有相似的精度。我们的数据表明，提取的碎片离子色谱图是DIA提供卓越重现性的原因。版权所有（c）2015 John Wiley＆Sons，Ltd.

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