A Surface Biotinylation Strategy for Reproducible Plasma Membrane Protein Purification and Tracking of Genetic and Drug-Induced Alterations
A Surface Biotinylation Strategy for Reproducible Plasma Membrane Protein Purification and Tracking of Genetic and Drug-Induced Alterations
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DOI：
10.1021/acs.jproteome.5b01066
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发表时间：
2016-02-01
影响因子：
4.4
通讯作者：
Bennettt, Keiryn L.
中科院分区：
文献类型：
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作者：
Hoermann, Katrin;Stukalov, Alexey;Bennettt, Keiryn L.
质膜(PM)蛋白有助于细胞的识别，介导接触和通讯，并占已知药物靶标的三分之二以上。(1-8)在过去的几年中，已经描述了几种PM蛋白质的蛋白质组学图谱。然而，比较分析主要集中在一种方法的不同变体上。(9-11)我们比较了磺基-NHS-SS-生物素化、氨氧基-生物素化和表面包被硅珠来分离PM蛋白，以便随后用一维无胶液色谱质谱进行分析。在定性和定量水平上评估PM蛋白质的绝对和相对数量以及重复性参数。在大多数监测标准中，磺化-NHS-SS-生物素化优于使用硅珠的氨氧基生物素化和表面涂层。我们通过竞争生物素洗脱策略进一步简化了这一过程，获得了PM注释的平均蛋白质组分54%(347种蛋白质)。使用其他数据库和预测工具进行的计算分析表明，总共超过90%的纯化蛋白与PM有关，主要是作为相互作用因子。通过跟踪遗传改变和药物治疗引起的质膜蛋白质组组成的变化，验证了改进的磺化-NHS-SS-生物素化方案。糖基磷脂酰肌醇(GPI)锚定的蛋白质在PM净化中被耗尽，缺乏GPI转氨酶成分的细胞，衣霉素处理的细胞显著减少了表面净化中N-糖蛋白的丰度。
Plasma membrane (PM) proteins contribute to the identity of a cell, mediate contact and communication, and account for more than two-thirds of known drug targets.(1-8) In the past years, several protocols for the proteomic profiling of PM proteins have been described. Nevertheless, comparative analyses have mainly focused on different variations of one approach.(9-11) We compared sulfo-NHS-SS-biotinylation, aminooxy-biotinylation, and surface coating with silica beads to isolate PM proteins for subsequent analysis by one-dimensional gel-free liquid chromatography mass spectrometry. Absolute and relative numbers of PM proteins and reproducibility parameters on a qualitative and quantitative level were assessed. Sulfo-NHS-SS-biotinylation outperformed aminooxy-biotinylation and surface coating using silica beads for most of the monitored criteria. We further simplified this procedure by a competitive biotin elution strategy achieving an average PM annotated protein fraction of 54% (347 proteins). Computational analysis using additional databases and prediction tools revealed that in total over 90% of the purified proteins were associated with the PM, mostly as interactors. The modified sulfo-NHS-SS-biotinylation protocol was validated by tracking changes in the plasma membrane proteome composition induced by genetic alteration and drug treatment. Glycosylphosphatidylinositol (GPI)-anchored proteins were depleted in PM purifications from cells deficient in the GPI transamidase component PIGS, and treatment of cells with tunicamycin significantly reduced the abundance of N-glycoproteins in surface purifications.