Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications
Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications
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DOI：
10.1093/protein/gzr054
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发表时间：
2012-01-01
影响因子：
2.4
通讯作者：
Barnes, Stephen
中科院分区：
文献类型：
--
作者：
McClure, Michelle;DeLucas, Lawrence J.;Barnes, Stephen
翻译后修饰（ptm）在许多跨膜蛋白的生物发生过程中起着至关重要的作用。此前，由于难以获得足够量的纯化蛋白，因此无法评估囊性纤维化跨膜传导调节剂（CFTR）中的PTMs， CFTR是囊性纤维化的上皮离子通道。我们最近使用了一种诱导过表达策略来产生适合纯化和详细分析的重组CFTR蛋白。利用液相色谱（LC）串联和多重反应离子监测（MRM）质谱，我们确定了PTMs的特定位点，包括棕榈酰化、磷酸化、甲基化和可能的泛素化。许多这些共价CFTR修饰以前没有被描述过，但可能影响关键和临床重要的分子过程，包括蛋白质成熟、门控和与疾病相关的某些突变的潜在机制。
Post-translational modifications (PTMs) play a crucial role during biogenesis of many transmembrane proteins. Previously, it had not been possible to evaluate PTMs in cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial ion channel responsible for cystic fibrosis, because of difficulty obtaining sufficient amounts of purified protein. We recently used an inducible overexpression strategy to generate recombinant CFTR protein at levels suitable for purification and detailed analysis. Using liquid chromatography (LC) tandem and multiple reaction ion monitoring (MRM) mass spectrometry, we identified specific sites of PTMs, including palmitoylation, phosphorylation, methylation and possible ubiquitination. Many of these covalent CFTR modifications have not been described previously, but are likely to influence key and clinically important molecular processes including protein maturation, gating and the mechanisms underlying certain mutations associated with disease.