An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes.
An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes.
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DOI：
10.1016/j.cels.2017.05.009
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发表时间：
2017-06-28
期刊：
影响因子：
9.3
通讯作者：
Olsen JV
中科院分区：
文献类型：
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作者：
Bekker-Jensen DB;Kelstrup CD;Batth TS;Larsen SC;Haldrup C;Bramsen JB;Sørensen KD;Høyer S;Ørntoft TF;Andersen CL;Nielsen ML;Olsen JV
本研究探讨了利用质谱（MS）为基础的散弹枪蛋白质组学对单细胞类型的完整人类蛋白质组进行全面编目的挑战。我们修改了一个经典的二维高分辨率反相肽分离方案，并优化了一个方案，提供足够的峰值容量，以饱和现代质谱仪器的测序速度。该策略实现了迄今为止人类单细胞类型最深的蛋白质组，HeLa蛋白质组测序深度为~ 584,000个独特肽序列和~ 14,200个蛋白质异构体（~ 12,200个蛋白质编码基因）。这种深度可与下一代RNA测序相媲美，并且无需富集即可识别翻译后修饰，包括~ 7,000个n -乙酰化位点和~ 10,000个磷酸化位点。我们通过全面量化几种不同人类癌细胞系和患者组织样本中的全球蛋白质组表达，进一步证明了这种蛋白质组学策略的一般适用性和临床潜力。多片段蛋白质组学定量了HeLa细胞中12200多个基因的蛋白质水平，这种基本完整的HeLa蛋白质组具有与下一代RNA-seq相似的覆盖范围，无需特异性富集即可实现对主要PTMs的深度覆盖。该方法可扩展到其他人类细胞系和患者样本，Bekker-Jensen等人表明，蛋白质组学现在可以提供基本完整的HeLa蛋白质组。他们同时提供超过12,200个蛋白质编码基因，10,000个磷酸化位点和7,000个n-乙酰化位点的测量。他们的方法快速，易于使用，并且需要少量的起始材料，这使得它很容易扩展到其他人类细胞系和患者样本。
This study investigates the challenge of comprehensively cataloging the complete human proteome from a single-cell type using mass spectrometry (MS)-based shotgun proteomics. We modify a classical two-dimensional high-resolution reversed-phase peptide fractionation scheme and optimize a protocol that provides sufficient peak capacity to saturate the sequencing speed of modern MS instruments. This strategy enables the deepest proteome of a human single-cell type to date, with the HeLa proteome sequenced to a depth of ∼584,000 unique peptide sequences and ∼14,200 protein isoforms (∼12,200 protein-coding genes). This depth is comparable with next-generation RNA sequencing and enables the identification of post-translational modifications, including ∼7,000 N-acetylation sites and ∼10,000 phosphorylation sites, without the need for enrichment. We further demonstrate the general applicability and clinical potential of this proteomics strategy by comprehensively quantifying global proteome expression in several different human cancer cell lines and patient tissue samples. Multi-shot proteomics quantifies the protein levels of 12,200+ genes in HeLa cells This essentially complete HeLa proteome has coverage similar to next-gen RNA-seq Deep coverage of major PTMs is achieved without specific enrichment The approach is extendable to other human cell lines and patient samples Bekker-Jensen et al. show that proteomics can now provide an essentially complete HeLa proteome. They provide measurements acquired simultaneously for more than 12,200 protein-coding genes, 10,000 phosphorylation sites, and 7,000 N-acetylation sites. Their approach is fast, accessible, and requires modest amounts of starting material, making it easily extendable to other human cell lines and patient samples.