PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry.
PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry.
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DOI：
10.1021/acs.jproteome.0c00303
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发表时间：
2020-09-04
影响因子：
4.4
通讯作者：
Takemori N
中科院分区：
文献类型：
--
作者：
Takemori A;Butcher DS;Harman VM;Brownridge P;Shima K;Higo D;Ishizaki J;Hasegawa H;Suzuki J;Yamashita M;Loo JA;Loo RRO;Beynon RJ;Anderson LC;Takemori N
来自生物样品的复杂蛋白质混合物的预折射对于通过自上而下的质谱（MS）进行蛋白质组分析是必不可少的。聚丙烯酰胺凝胶电泳（PAGE）能够根据分子大小进行高分辨率蛋白质分离，是生物化学实验中广泛使用的技术，并且有可能用于自上而下的MS分析的样品分级。然而，缺乏一种手段，以有效地回收分离的蛋白质在凝胶一直是一个障碍，它的使用在样品预分馏。在这项研究中，我们提出了一种新的实验工作流程，称为被动洗脱蛋白质从聚丙烯酰胺凝胶作为完整的物种MS（'PEPPI-MS'），它允许自上而下的PAGE分离的蛋白质的MS。在PEPPI-MS工作流程中优化考马斯亮蓝染色，然后进行被动提取步骤，能够在10分钟内从广泛的分子量区域有效回收在商业预制凝胶上分离的蛋白质。结合离线PEPPI-MS和在线反相液相色谱分离的二维分离导致从凝胶的目标区域（≤50 kDa）回收的超过1000种蛋白质型的鉴定。鉴于传统SDS-PAGE设备的广泛可用性和相对较低的成本，PEPPI-MS工作流程将是自上而下的蛋白质组学的强大的预分离策略。
Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS (‘PEPPI-MS’), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie Brilliant Blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 minutes. Two-dimensional separation combining off-line PEPPI-MS with on-line reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional SDS-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.
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