Plasma membrane proteins perform a variety of important tasks in the cells. These tasks can be diverse as carrying nutrients across the plasma membrane, receiving chemical signals from outside the cell, translating them into intracellular action, and anchoring the cell in a particular location. When these crucial roles of plasma membrane proteins are considered, the need for their characterization becomes inevitable. Certain characteristics of plasma membrane proteins such as hydrophobicity, low solubility, and low abundance limit their detection by proteomic analyses. Here, we presented a comparative proteomics study in which the most commonly used plasma membrane protein enrichment methods were evaluated. The methods that were utilized include biotinylation, selective CyDye labeling, temperature-dependent phase partition, and density-gradient ultracentrifugation. Western blot analysis was performed to assess the level of plasma membrane protein enrichment using plasma membrane and cytoplasmic protein markers. Quantitative evaluation of the level of enrichment was performed by two-dimensional electrophoresis (2-DE) and benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE) from which the protein spots were cut and identified. Results from this study demonstrated that density-gradient ultracentrifugation method was superior when coupled with 16-BAC/SDS-PAGE. This work presents a valuable contribution and provides a future direction to the membrane sub-proteome research by evaluating commonly used methods for plasma membrane protein enrichment.

中文翻译：

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四种常用方法鉴定新型血浆膜蛋白的比较蛋白质组学分析。

质膜蛋白在细胞中执行多种重要任务。这些任务可能是多种多样的，例如，将营养物携带通过质膜，从细胞外部接收化学信号，将其转化为细胞内作用以及将细胞锚定在特定位置。当考虑质膜蛋白的这些关键作用时，对其表征的需求变得不可避免。质膜蛋白的某些特性（例如疏水性，低溶解度和低丰度）限制了它们通过蛋白质组学分析的检测。在这里，我们提出了一个比较蛋白质组学研究，其中评估了最常用的质膜蛋白富集方法。使用的方法包括生物素化，选择性CyDye标记，温度依赖性相分配，和密度梯度超速离心。使用质膜和细胞质蛋白标记物进行蛋白质印迹分析以评估质膜蛋白富集水平。通过二维电泳（2-DE）和苄基二甲基-正十六烷基氯化铵/十二烷基硫酸钠聚丙烯酰胺凝胶电泳（16-BAC / SDS-PAGE）进行富集水平的定量评估，从中切出蛋白质斑点，确定。这项研究的结果表明，与16-BAC / SDS-PAGE结合使用密度梯度超速离心法效果更好。这项工作提出了宝贵的贡献，并通过评估常用的质膜蛋白富集方法为膜子蛋白质组学研究提供了未来的方向。使用质膜和细胞质蛋白标记物进行蛋白质印迹分析以评估质膜蛋白富集水平。通过二维电泳（2-DE）和苄基二甲基-正十六烷基氯化铵/十二烷基硫酸钠聚丙烯酰胺凝胶电泳（16-BAC / SDS-PAGE）进行富集水平的定量评估，从中切出蛋白质斑点，确定。这项研究的结果表明，与16-BAC / SDS-PAGE结合使用密度梯度超速离心法效果更好。这项工作提出了宝贵的贡献，并通过评估常用的质膜蛋白富集方法为膜子蛋白质组学研究提供了未来的方向。使用质膜和细胞质蛋白标记物进行蛋白质印迹分析以评估质膜蛋白富集水平。通过二维电泳（2-DE）和苄基二甲基-正十六烷基氯化铵/十二烷基硫酸钠聚丙烯酰胺凝胶电泳（16-BAC / SDS-PAGE）进行富集水平的定量评估，从中切出蛋白质斑点，确定。这项研究的结果表明，与16-BAC / SDS-PAGE结合使用密度梯度超速离心法效果更好。这项工作提出了宝贵的贡献，并通过评估常用的质膜蛋白富集方法为膜子蛋白质组学研究提供了未来的方向。