Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using nonphosphorylated and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC-MS. Separate LC-MS analyses of Lys-C, Lys-C plus trypsin, and Lys-C plus Glu-C digests provided 100% sequence coverage, including the identification of a very large 13 kDa Lys-C fragment using a special sample handling procedure (4 M guanidine HCl) prior to the LC-MS analysis to improve the large peptide recovery. The analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser15, enabled quantitative stoichiometry of Hsp27 phosphorylation by LC-MS. The strategy revealed details of Hsp27 phosphorylation, including significant di-phosphorylation at both Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipation, and LC-MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the antiangiogenic activities of thiolutin and related dithiolethiones.
硫藤黄菌素是微生物的一种硫基复合物，已知具有血管生成抑制剂的活性。相对于以前研究的血管生成抑制剂，硫藤黄菌素是一个非常强的热休克蛋白27（HSP 27）磷酸化的诱导剂。这种磷酸化作用需要p38激酶，但与p38激酶的促磷化作用是独立的。为阐明硫藤黄菌素如何调节热休克蛋白的磷酸化，并最终促进血管生成，将人脐静脉内皮细胞用硫藤黄菌素处理或不用处理，细胞裂解后提取HSP 27，用非磷酸化和磷酸化热休克蛋白27抗体(Phospho-Ser78)来免疫共沉淀。以赖氨酸-C肽链内切酶、赖氨酸-C肽链内切酶+胰蛋白酶、赖氨酸-C肽链内切酶+谷氨酸-C肽链内切酶消化后，用液相色谱来分析，以获取100%的序列覆盖范围。非常大的13KD的赖氨酸-C片段，在进行液相色谱分析前，用一种特殊的试剂（4M的盐酸胍）来处理，以提高大片段的恢复。用液相色谱定量化学计量学分析揭示了一个新的HSP27翻译后修饰：截去N端的蛋氨酸和倒数第二位的苏氨酸乙酰化。谷氨酸C端内切后片段分析后发现两个磷化位点，丝氨酸78和丝氨酸82，胰蛋白酶片段显示另一个磷酸化位点，丝氨酸15。本研究策略揭示了HSP27磷酸化的细节，包括在丝氨酸78和丝氨酸82位的双磷酸化。这些研究很难用传统的方法获得，因为疏水性N端区域的寡聚化防止了高效的酶切分析。western印迹、免疫沉淀和液相色谱相结合提供了硫藤黄菌素刺激HSP27磷酸化的定量分析，并且进一步描述了HSP27对硫藤黄菌素及其相关的二硫基乙硫酸在抗血管生成中的活性的作用。
