Genome-specific gas-phase fractionation strategy for improved shotgun proteomic profiling of proteotypic peptides
Genome-specific gas-phase fractionation strategy for improved shotgun proteomic profiling of proteotypic peptides
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DOI：
10.1021/ac701680f
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发表时间：
2008-02-15
影响因子：
7.4
通讯作者：
Goodlett, David R.
中科院分区：
文献类型：
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作者：
Scherl, Alexander;Shaffer, Scott A.;Goodlett, David R.
气相分级分离 (GPF) 是一种提高蛋白质组覆盖率的有效且简单的方法。在本报告中，最佳 m/z 范围是根据基因组复杂性和实验数据计算的。然后，从各种生物体的基因组中通过计算机计算理论母离子密度，并发现证实了基于离子密度图的 m/z 范围的经验选择。根据这两种计算，在较低 m/z 范围内最有效 GPF 覆盖的 m/z 范围的选择应该非常窄，并且随着 m/z 值的增加而增加。接下来，进行系统的 LC-MS/MS 分析以证实这一观察结果。在 LTQ-Orbitrap 混合质谱仪的三种不同扫描模式下研究了数据依赖性母离子选择的行为和观察到的变异性的起源。最后，将 GPF 与数据依赖分析相结合，与基于 LC-ESI-MS/MS 数据的经验观察的蛋白质肽的靶向伪多反应监测分析进行比较，该分析应该是可检测的。后一个实验的结果支持了我们的结论，即使用合理气相分级的数据依赖性分析足以对未分级的细胞裂解物中的蛋白质型肽进行全面的蛋白质组分析。
Gas-phase fractionation (GPF) is an efficient and straightforward method to increase proteome coverage. In this report, optimal m/z ranges were calculated based on genomic complexity and experimental data. Then, theoretical precursor ion densities were calculated in silico from various organisms' genomes and found to corroborate the empirical selection of m/z ranges based on ion density mapping. According to both calculations, the choice of m/z range for most efficient GPF coverage in the lower m/z range should be very narrow and increase as m/z value increases. Next, a systematic LC-MS/MS analysis was performed to confirm this observation. The behavior of data-dependent precursor ion selection and the origin of the observed variabifity was investigated under three different scan modes of an LTQ-Orbitrap hybrid mass spectrometer. Finally, GPF combined with data-dependent analysis was compared to a targeted, pseudo-multiple reaction monitoring analysis of proteotypic peptides that should be, based on empirical observation of LC-ESI-MS/MS data, detectable. The result of the latter experiment supported our conclusion that data-dependent analysis using rational gas-phase fractionation was sufficient for comprehensive proteomic analysis of the proteotypic peptides in an unfractionated cell lysate.