Mass spectrometry of phosphatidylinositols with various fatty acid side chain

磷脂酰肌醇3-激酶（Phosphatidylinositol 3-kinase, Pl 3-kinase）是3-磷酸化多磷酸肌醇信号通路中的关键酶。在体内，pl3 -激酶的主要底物被认为是磷脂酰肌醇4,5 -二磷酸（pl4,5 - p_2），产生pl3,4,5 - p_3，这是一个被认为在信号转导中起重要作用的第二信使。在体外，该酶能磷酸化Pl、Pl 4- p和Pl 4,5 - p_2，从动物组织中纯化的天然Pl是Pl 3-激酶的良好底物，在sn-2位置含有花生四烯酸酯。另一方面，我们发现在sn-2位置具有18号和20号碳的饱和脂肪酸的合成Pls没有被酶磷酸化。为了评价与Pl 3-激酶的磷酸化反应，我们对Pl的sn-2脂肪酸进行了优化，以7种羧酸（C4、C6、C8、C10、C10、C12、C14、C16）为原料，在sn-2中心合成了具有多种饱和脂肪酸的Pl （Pl_<C4>、Pl_<C6>、Pl_<C8>、Pl_<C10>、Pl_<C12>、Pl_<C14>、Pl_<C16>）混合物，并与Pl 3-激酶进行酶促反应。采用负离子快原子轰击（FAB）质谱法，以三乙醇胺：甘油= 3:1为基质进行分析。光谱显示单磷酸Pl_<C4>、Pl_<C6>、Pl_<C8>和Pl_<C10>类似物的[M-H]离子峰。较长链类似物如Pl_<C16>的磷酸化离子仅在痕量水平上检测到。短的sn-2脂肪酸类似物如Pl_<C4>, Pl_<C8>也被证明是pl3 -激酶的优良底物，通过与r3 -激酶和[γ -^<32>P]- atp独立磷酸化合成的Pls （Pl_<C2>, Pl_<C4>, Pl_<C8>, Pl_<C12>, Pl_<C16>, Pl_<C18>）。这些结果可用于人工第二信使分子、酶抑制剂、寻找特异性结合蛋白的亲和探针和光亲和标记探针的设计。

Phosphatidylinositol 3-kinase (Pl 3-kinase) is a key enzyme in the signaling pathways of 3-phosphorylated polyphosphoinositides. The major substrate of Pl 3-kinase, in vivo, is assumed to be phosphatidylinositol 4, 5-bisphosphate (Pl 4, 5-P_2), generating Pl 3, 4, 5-P_3, a putative second messenger to play important roles in signal transduction. In vitro, the enzyme phosphorylates Pl, Pl 4-P and Pl 4, 5-P_2, The natural Pl purified from animal tissues, which is an excellent substrate of Pl 3-kinase, contains arachidonate at the sn-2 position. On the other hand, we found that synthetic Pls with saturated fatty acid of carbon number 18 and 20 at the sn-2 position, were not phosphorylated by the enzyme. In order to evaluate the phosphorylation reaction with Pl 3-kinase, we optimized the sn-2 fatty acids of Pl.A mixture of Pls (Pl_<C4>, Pl_<C6>, Pl_<C8>, Pl_<C10>, Pl_<C12>, Pl_<C14>, Pl_<C16>) with various saturated fatty acids at the sn-2 center was synthesized from an equimolar mixture of seven carboxylic acids (C4, C6, C8, C10, C12, C14, C16), and was subjected to the enzymatic reaction with Pl 3-kinase. The product was analyzed by negative ion fast atom bombardment (FAB) mass spectrometry using a matrix (triethanolamine : glycerol = 3 : 1). The spectrum showed the [M-H] ion peaks of monophosphorylated Pl_<C4>, Pl_<C6>, Pl_<C8> and Pl_<C10> analogs. The phosphorylated ions derived from the longer chain analogs such as Pl_<C16> were detected only at trace levels. Short sn-2 fatty acid analogs such as Pl_<C4>, Pl_<C8> were also shown to be excellent substrates of Pl 3-kinase by independent phosphorylation of synthetic Pls (Pl_<C2>, Pl_<C4>, Pl_<C8>, Pl_<C12>, Pl_<C16>, Pl_<C18>) with Rl 3-kinase and [gamma-^<32>P]-ATP.These results are applicable for the design of artificial second messenger molecules, enzyme inhibitors, affinity probes to find specific binding proteins and photoaffinity labeling probes.