DIA-SIFT: A Precursor and Product Ion Filter for Accurate Stable Isotope Data-Independent Acquisition Proteomics
DIA-SIFT: A Precursor and Product Ion Filter for Accurate Stable Isotope Data-Independent Acquisition Proteomics
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DOI：
10.1021/acs.analchem.8b01618
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发表时间：
2018-08-07
影响因子：
7.4
通讯作者：
Martin, Brent R.
中科院分区：
文献类型：
--
作者：
Haynes, Sarah E.;Majmudar, Jaimeen D.;Martin, Brent R.
基于定量质谱的蛋白质谱分析被广泛用于测量不同治疗或疾病状态下的蛋白质水平，但目前的质谱采集方法存在明显的局限性。虽然数据独立采集（DIA）绕过了数据依赖采集（DDA）的随机性，但从DIA获得的碎片光谱通常很复杂，并且难以去卷积。在线离子迁移率分离（IMS）增加了一个额外的维度，以增加更有效的产物离子分配的峰容量。作为一个类似的策略，顺序窗口采集方法（SWATH），IMS使能的DIA方法竞争DDA方法的蛋白质注释。在这里，我们评估IMS-DIA定量准确性使用稳定同位素标记的氨基酸在细胞培养（SILAC）。由于SILAC分析使样品复杂性加倍，我们发现IMS-DIA分析对于灵敏定量不够准确。然而，SILAC前体对共享共同的保留和漂移时间，并且两种物质共片段化以产生多个可定量的同位素y离子峰对。由于y-离子SILAC比率是每个定量前体的固有比率，因此组合的MS 1和y-离子比率分析显著增加了测量的总数。随着采样的增加，我们提出了DIA-SIFT（SILAC内在过滤工具），一个简单的统计算法，以识别和消除量化不佳的MS 1和/或MS 2事件。DIA-SIFT结合MS 1和y离子比率，去除离群值，并提供更准确和精确的定量（
Quantitative mass spectrometry-based protein profiling is widely used to measure protein levels across different treatments or disease states, yet current mass spectrometry acquisition methods present distinct limitations. While data-independent acquisition (DIA) bypasses the stochastic nature of data-dependent acquisition (DDA), fragment spectra derived from DIA are often complex and challenging to deconvolve. In-line ion mobility separation (IMS) adds an additional dimension to increase peak capacity for more efficient product ion assignment. As a similar strategy to sequential window acquisition methods (SWATH), IMS-enabled DIA methods rival DDA methods for protein annotation. Here we evaluate IMS-DIA quantitative accuracy using stable isotope labeling by amino acids in cell culture (SILAC). Since SILAC analysis doubles the sample complexity, we find that IMS-DIA analysis is not sufficiently accurate for sensitive quantitation. However, SILAC precursor pairs share common retention and drift times, and both species cofragment to yield multiple quantifiable isotopic y-ion peak pairs. Since y-ion SILAC ratios are intrinsic for each quantified precursor, combined MS1 and y-ion ratio analysis significantly increases the total number of measurements. With increased sampling, we present DIA-SIFT (SILAC Intrinsic Filtering Tool), a simple statistical algorithm to identify and eliminate poorly quantified MS1 and/or MS2 events. DIA-SIFT combines both MS1 and y-ion ratios, removes outliers, and provides more accurate and precise quantitation (