Mass spectrometry of phosphatidylinositols with various fatty acid side chain

磷脂酰肌醇3-激酶（PI 3-kinase）是3-磷酸化多聚磷酸肌醇信号通路中的关键酶。P13-激酶在体内的主要底物是磷脂酰肌醇4，5-二磷酸（P14，5-P_2），产生P13，4，5-P_3，P13，4，5-P_3被认为是信号转导中的第二信使。从动物组织中纯化的天然磷脂是磷脂酰肌醇3-激酶的优良底物，其sn-2位含有花生四烯酸。另一方面，我们发现在sn-2位具有碳数18和20的饱和脂肪酸的合成磷脂不被该酶磷酸化。为了研究磷脂酰肌醇3-激酶对磷脂酰肌醇3-激酶磷酸化反应的影响，我们对磷脂酰肌醇3-激酶的sn-2位脂肪酸进行了优化。以等摩尔的<C4><C6><C8><C10><C12><C14><C16>7种羧酸（C4，C6，C8，C10，C12，C14，C16）混合物为原料，合成了在sn-2位含有各种饱和脂肪酸的磷脂酰肌醇3-激酶混合物（Pl_，Pl_，Pl_，Pl_，Pl_，Pl_，Pl_），并与磷脂酰肌醇3-激酶进行了酶促反应。使用基质（三乙醇胺：甘油= 3：1）通过负离子快原子轰击（FAB）质谱法分析产物。光谱显示单磷酸化的Pl_<C4>、Pl_<C6>、Pl_<C8>和Pl_<C10>类似物的[M-H]离子峰。仅在痕量水平检测到源自较长链类似物如PL的磷酸化离子<C16>。短sn-2脂肪酸类似物如Pl_<C4>，Pl_<C8>是合成的Pl_（Pl_<C2>，Pl_，Pl<C4>_，Pl_<C8>，Pl<C12>_，<C16>Pl_<C18>）与Rl 3-激酶和[γ-^<32>P]-ATP的独立磷酸化反应也显示出Pl 3-激酶的优良底物，这些结果可用于人工第二信使分子、酶抑制剂、寻找特异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.