Schebb Nils Helge, Falck David, Faber Helene, Hein Eva-Maria, Karst Uwe, Hayen Heiko
Westfälische Wilhelms-Universität Münster, Institut für Anorganische und Analytische Chemie, Corrensstrasse 30, 48149 Münster, Germany.
J Chromatogr A. 2009 Jul 3;1216(27):5249-55. doi: 10.1016/j.chroma.2009.05.020. Epub 2009 May 18.
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 microm 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 microm) 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.
已开发出一种用于快速测定磷脂酶A(2)（PLA(2)）活性的新型液相色谱 - 电喷雾电离质谱（LC - ESI - MS）方法。该方法首次实现了对作为PLA(2)底物的甘油磷脂酰胆碱（GroPCho）及其产物脂肪酸（FA）和溶血 - GroPCho的并行检测。ESI - MS在负离子模式下进行，将FA检测为M - H离子，将溶血 - GroPCho和GroPCho检测为乙酸盐加合物M + Ac。在具有3微米颗粒的短C(5)改性硅胶柱上采用快速梯度洗脱，五个GroPChos、五个FAs和六个溶血 - GroPChos能够在不到3分钟的时间内根据其链长分离。分离GroPChos时实现了非常高的平均色谱效率，达41,200理论塔板数（塔板高度0.5微米）。该方法用于监测花生四烯酸（20:4 FA）和1 - 硬脂酰 - 溶血 - sn - GroPCho（18:0 GroPCho）从1 - 硬脂酰 - 2 - 花生四烯酰 - sn - GroPCho（18:0/20:4 GroPCho）单层囊泡中的释放。对于FAs和溶血 - GroPChos，检测限为柱上进样总量0.5皮摩尔，对于GroPChos为1.5皮摩尔，线性范围为1.5个数量级，该方法已证明适用于监测不同分泌型PLA(2)（sPLA(2)）的转化。此外，它还用于筛选一小批PLA(2)抑制剂对V型sPLA(2)和莫氏矛头蝮蛇毒的活性。在这两种情况下，均可直接鉴定出活性样品。所开发的LC - ESI - MS方法分析时间短、色谱效率高且能并行检测底物和所有产物，非常适合用于分析PLA(2)活性。
