Recent advances in mass spectrometry and separation technologies created the opportunities for deep proteome characterization using shotgun proteomics approaches. The “real world” sample complexity and high concentration range limit the sensitivity of this characterization. The common strategy for increasing the sensitivity is sample fractionation prior to analysis either at the protein or the peptide level. Typically, fractionation at the peptide level is performed using linear gradient high-performance liquid chromatography followed by uniform fraction collection. However, this way of peptide fractionation results in significantly suboptimal operation of the mass spectrometer due to the non-uniform distribution of peptides between the fractions. In this work, we propose an approach based on peptide retention time prediction allowing optimization of chromatographic conditions and fraction collection procedures. An open-source software implementing the approach called FractionOptimizer was developed and is available at http://hg.theorchromo.ru/FractionOptimizer. The performance of the developed tool was demonstrated for human embryonic kidney (HEK293) cell line lysate. In these experiments, we improved the uniformity of the peptides distribution between fractions. Moreover, in addition to 13,492 peptides, we found 6787 new peptides not identified in the experiments without fractionation and up to 800 new proteins (or 25%).

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质谱和分离技术的最新进展为使用shot弹枪蛋白质组学方法进行深层蛋白质组表征创造了机会。“真实世界”的样品复杂性和高浓度范围限制了该表征的灵敏度。提高灵敏度的常用策略是在蛋白质或肽水平上进行分析之前对样品进行分级分离。通常，使用线性梯度高效液相色谱法在肽水平上进行分馏，然后进行均匀的馏分收集。但是，由于肽在组分之间的分布不均匀，因此这种肽分离方法会导致质谱仪的操作明显欠佳。在这项工作中，我们提出了一种基于肽保留时间预测的方法，该方法可以优化色谱条件和馏分收集程序。开发了一种实现该方法的开源软件，称为FractionOptimizer，该软件可从以下网站获得：http://hg.theorchromo.ru/FractionOptimizer。该开发工具的性能已证明可用于人胚胎肾（HEK293）细胞系裂解液。在这些实验中，我们改善了各部分之间肽分布的均匀性。此外，除了13,492个肽段外，我们发现实验中未鉴定的6787个新肽段没有进行分离，并且多达800个新蛋白（或25％）。

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