Proteogenomic Analyses of Cellular Lysates Using a Phenol-Guanidinium Thiocyanate Reagent
Proteogenomic Analyses of Cellular Lysates Using a Phenol-Guanidinium Thiocyanate Reagent
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DOI：
10.1021/acs.jproteome.8b00609
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发表时间：
2019-01-01
影响因子：
4.4
通讯作者：
Ohara, Osamu
中科院分区：
文献类型：
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作者：
Kawashima, Yusuke;Miyata, Jun;Ohara, Osamu
在全面了解复杂生物系统的动态时，蛋白质和 RNA 谱可以提供丰富的信息。蛋白质和 RNA 谱之间的定量相关性并不总是很高，但同时获取这两种谱仍然具有挑战性，部分原因是样品的可用性有限以及单独制备蛋白质和 RNA 组分的不便。在之前的一项研究中，使用异硫氰酸酚胍试剂 (P/GTC) 从同一样品中同时制备蛋白质、DNA 和 RNA 组分，尽管 P/GTC 提取物在蛋白质组分析中的性能仍知之甚少。因此，我们使用标准 HEK293-F 细胞和人外周血中性粒细胞评估了 P/GTC 提取方法在蛋白质组分析中的性能。后一种细胞类型因其极易遭受蛋白质/RNA 降解而闻名，这反映了较高的蛋白酶和核糖核酸酶活性。我们的数据表明，与传统的蛋白质提取方法相比，P/GTC 提取方法可以从中性粒细胞和 HEK293-F 细胞样品中同时制备 RNA 和蛋白质，从而提供优异的蛋白质谱。因此，P/GTC 提取方法为广泛的蛋白质组学研究提供了强大而稳健的工具。
Protein and RNA profiles are highly informative when generating a comprehensive understanding of the dynamics of complex biological systems. Quantitative correlations between protein and RNA profiles are not always high, yet simultaneous acquisition of both profiles remains challenging, in part because of the limited availability of samples and the inconvenience of separately preparing protein and RNA fractions. In a previous study, protein, DNA, and RNA fractions were simultaneously prepared from the same sample using phenol-guanidinium isothiocyanate reagent (P/GTC), although the performance of P/GTC-extracts in proteogenomic analyses remains poorly understood. We therefore evaluated the performance of the P/GTC-extraction method in proteogenomic analyses using standard HEK293-F cells and human peripheral blood neutrophils. The latter cell type is renowned for its extreme vulnerability to protein/RNA degradation, reflecting high protease and RNase activities. Our data indicate that the P/GTC extraction method provides superior protein profiles from neutrophil and HEK293-F cell samples for simultaneous preparation of RNA and protein, as compared with those from conventional protein extraction methods. The P/GTC extraction method therefore provides a powerful and robust tool for a broad range of proteogenomic studies.