Department of Basic Medical Sciences, Medical College of Taizhou University, Taizhou, Zhejiang 318000, China.
Protein J. 2010 Nov;29(8):531-7. doi: 10.1007/s10930-010-9285-x.
Research has shown that the palmitoyl group of α-tubulin mediates the hydrophobic interaction between microtubules and intracellular membranes and that palmitoylated tubulin plays a role in signal transduction. There are 20 cysteine residues per α/β tubulin heterodimer. C376 of α-tubulin was reported to be predominantly palmitoylated and C20, C213 and C305 of α-tubulin were palmitoylated at lower levels. The previous method used for the analysis of the palmitoylation sites on α-tubulin was based on ³H-labeling, enzymolysis, purification and sequencing. This approach, although efficient, is laborious. Mass spectrometry (MS), especially tandem MS, has been shown to be a successful method for identification of various post-translational modifications of proteins. We report here a convenient MS-based method to comprehensively analyze the palmitoylation sites of the α/β tubulin heterodimer. Acyl-biotinyl exchange chemistry and streptavidin agarose affinity purification were applied to enrich palmitoylated peptides from tubulin. After nano-LC-MS/MS analysis, database searching and manual analysis of the spectra revealed that 11 cysteine residues of the α/β tubulin heterodimer were palmitoylated.
研究表明，α-微管蛋白的棕榈酰基介导了微管和细胞内膜之间的疏水相互作用，并且棕榈酰化的微管蛋白在信号转导中发挥作用。每个α/β微管蛋白异二聚体有 20 个半胱氨酸残基。据报道，α-微管蛋白的 C376 主要被棕榈酰化，而 C20、C213 和 C305 的棕榈酰化水平较低。以前用于分析α-微管蛋白棕榈酰化位点的方法基于 ³H 标记、酶解、纯化和测序。虽然这种方法效率高，但很繁琐。质谱（MS），特别是串联 MS，已被证明是鉴定蛋白质各种翻译后修饰的成功方法。我们在这里报告了一种基于 MS 的方便方法，可全面分析α/β微管蛋白异二聚体的棕榈酰化位点。酰基辅氨酸交换化学和链霉亲和素琼脂糖亲和纯化用于从微管蛋白中富集棕榈酰化肽。经过纳升液相色谱-串联质谱（nano-LC-MS/MS）分析、数据库搜索和谱图手动分析，发现α/β微管蛋白异二聚体的 11 个半胱氨酸残基被棕榈酰化。
