光化学反应-串联质谱法鉴定细胞中不饱和卵磷脂双键的位置

以二苯甲酮(Benzophenone,BP)为光化学反应的反应物,建立了乳腺细胞中不饱和卵磷脂双键位置和双键异构体相对含量的在线光化学反应-脉冲直流电喷雾串联质谱(PB-pulsed-dc-ESI-MS/MS)的检测方法.通过三相萃取法提取细胞中的脂类,在254 nm紫外光的照射下,使不饱和卵磷脂中的C詤C和BP中的羰基发生特异性的环加成反应(Paternó-Bǜchi,PB反应),将反应后的PB产物通过非接触式的脉冲直流电喷雾(Pulsed directed current electrospray,pulsed-dc-ESI)方式电离,并进行低能碰撞裂解得到二级串联质谱(MS/MS),从得到的二级谱图的子离子中可以获得双键位置以及双键异构体相对含量的信息.检测了癌细胞MCF-7和正常细胞MCF-10A中8种不饱和卵磷脂中的双键位置,且对其中4种双键异构体的相对含量进行分析,发现PC 16:018:1中△9异构体的含量在两种细胞中差异不明显,PC 18:018:1和PC 18:118:1中△9异构体的含量略有差异,LPC 18:1中△9异构体的含量在癌细胞和正常两种细胞中差异比较明显(56.0%±1.3%vs.71.7%±6.8%).本方法快速简易,可用于分析双键位置及双键异构体相对含量,有望对不同细胞状态以及不同疾病状态进行鉴别.

Identification of C C Location of Unsaturated Phosphatidylcholines in Cell by Photochemical Reaction-Tandem Mass Spectrometry

A method of identification of C=C location and relative quantitation of unsaturated phosphatidylcholine ( PC ) isomers in breast cells by online photochemical reaction-pulsed directed current electrospray-tandem mass spectrometry ( PB-pulsed-dc-ESI-MS/MS) was established with benzophenone ( BP) as a photochemical reactant. The three-phase extraction method was used to extract the lipids in the cells, and then the C=C in the unsaturated PC and the carbonyl in BP were specifically cycled under the irradiation of 254 nm ultraviolet light (Paternò-Büchi, PB reaction). The PB products were ionized and mass-isolated for low-energy collision dissociation through the non-contact pulsed-dc-ESI ionization method. The double bond position and the relative content of the location isomers were obtained from the resulting ions in the MS/MS spectrum. The C=C location of 8 kinds of unsaturated PCs in MCF-7 and MCF-10A was detected, and the relative contents of 4 kinds of C=C location isoforms were analyzed. It was found that the relative abundance of △9 isomer in PC 16:018:1 was not significantly different between the two cells. The relative abundance of△9 isomers in PC 18:018:1 and PC 18:118:1 was slightly different. However, there is a big difference of △9 in LPC 18:1 between the cancer cell and normal cell (56. 0% ± 1. 3% vs. 71. 7% ± 6. 8%). The establishment of such a rapid and easy mass spectrometry method can analyze the C=C location and the relative content of location isomers, and it is expected to be a powerful tool to identify different cell states and different disease states.