Fast method for monitoring phospholipase A2 activity by liquid chromatography-electrospray ionization mass spectrometry
Fast method for monitoring phospholipase A2 activity by liquid chromatography-electrospray ionization mass spectrometry
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DOI：
10.1016/j.chroma.2009.05.020
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发表时间：
2009-07-03
影响因子：
4.1
通讯作者：
Hayen, Heiko
中科院分区：
文献类型：
--
作者：
Schebb, Nils Helge;Falck, David;Hayen, Heiko
建立了一种快速测定磷脂酶A（2）（PLA（2））活性的液相色谱-电喷雾电离质谱（LC-ESI-MS）新方法。该方法首次允许并行检测作为PLA（2）底物的甘油磷脂酰胆碱（GroPCho）及其产物脂肪酸（FA）和lyso-GroPCho。以负离子模式进行ESI-MS，检测FA为[M-H]（-）离子，检测溶血-GroPCho和GroPCho为乙酸盐加合物[M + Ac]（-）。在短C-5改性硅胶柱上利用快速梯度，3 μ m颗粒，5个GroPChos。根据它们的链长，可以在不到3分钟的时间内分离5种FA和6种溶血-GroPChos。对于GroPChos的分离，实现了41，200个理论塔板（塔板高度0.5 μ m）的非常高的平均色谱效率。该方法用于监测花生四烯酸（20：4 FA）和1-硬脂酰-lyso-sn-GroPCho（18：0 GroPCho）从1-硬脂酰-2-花生四烯酸-sn-GroPCho（18：0/20：4 GroPCho）单层囊泡中的释放。FA和lyso-GroPChos的检测限为0.5 pmol（柱上进样总量），GroPChos的检测限为1.5 pmol，线性范围为1.5十倍，该方法已被证明适用于监测不同的分泌型PLA（2）（sPLA（2））转化率。此外，它被应用于筛选PLA（2）抑制剂的小文库，用于它们对sPLA（2）V型和Bothrops moojeni蛇毒的活性。在这两种情况下，可以直接识别活性样品。LC-ESI-MS法分析时间短，色谱效率高，底物和产物可同时检测，适用于PLA（2）活性的分析。(C)2009 Elsevier B. V.保留所有权利。
A new liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for the fast determination of phospholipase A(2) (PLA(2)) activity has been developed. For the first time, the method allows the parallel detection of glycerophosphatidylcholine (GroPCho) as PLA(2) substrate as well as of its products fatty acid (FA) and lyso-GroPCho. ESI-MS was carried out in negative ion mode, detecting the FA as [M - H](-) ions and the lyso-GroPCho and GroPCho as acetate adducts [M + Ac](-). Utilizing a fast gradient on a Short C-5-modified silica gel column with 3 mu m particles, five GroPChos. five FAs and six lyso-GroPChos could be separated according to their chain length in less than 3 min. A very high average chromatographic efficiency of 41,200 theoretical plates (plate height 0.5 mu m) was achieved for the separation of the GroPChos. The method was applied for monitoring the release of arachidonic acid (20:4 FA) and 1-stearoyl-lyso-sn-GroPCho (18:0 GroPCho) from unilamellar vesicles of 1-stearoyl-2-arachidonoyl-sn-GroPCho, (18:0/20:4 GroPCho). With a limit of detection of 0.5 pmol (total amount injected on column) for the FAs and lyso-GroPChos and 1.5 pmol for the GroPChos as well as a linear range of 1.5 decades, the method has proven to be suitable for the monitoring of different secretory PLA(2) (sPLA(2)) conversions. Furthermore, it was applied to screen a small library of PLA(2) inhibitors for their activity towards sPLA(2) type V and snake venom of Bothrops moojeni. In both cases, active samples could be directly identified. With its short analysis time, its high chromatographic efficiency and the parallel detection of substrate and all products, the developed LC-ESI-MS method is well suited for the analysis of PLA(2) activity. (C) 2009 Elsevier B.V. All rights reserved.