Liu Shuqing, Zhang Cuili, Xu Yue-Fei, Yang Fan, Sun Ming-Zhong
Department of Biochemistry & Molecular Biology, Dalian Medical University, Dalian, 116044, China.
Rapid Commun Mass Spectrom. 2009 Apr;23(8):1158-66. doi: 10.1002/rcm.3996.
Results from high-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry (HPLC/nESI-MS/MS) coupled to two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE) indicated that the monomer and dimer of phospholipase A(2) (PLA(2)) coexisted in crude Chinese Agkistrodon blomhoffii Ussurensis snake venom (ABUSV). Then, an acidic PLA(2) with the accurate molecular mass of 13979.6 Da was purified from ABUSV (mo-ABUSV-aPLA(2)). MS/MS-derived peptides from ABUSV-aPLA(2) were compared with other homologous snake venom PLA(2)s, which in turn showed that ABUSV-aPLA(2) is a novel snake venom PLA(2). Meanwhile, the ABUSV-aPLA(2) dimer (di-ABUSV-aPLA(2)) was also obtained. MS/MS analysis identified the same peptides from di-ABUSV-aPLA(2) as from mo-ABUSV-aPLA(2), which indicates that di-ABUSV-aPLA(2) is a homodimer. One Ca(2+) ion is contained per ABUSV-aPLA(2). The Ca(2+) ion is critical for both the hydrolytic activity and the structure of ABUSV-aPLA(2). Pro-Q Emerald and Pro-Q Diamond specific glycoprotein and phosphoprotein staining combined with MS/MS analysis indicated that the ABUSV-aPLA(2) is both a glycoprotein and a phosphoprotein, which to our knowledge is the first such report for a snake venom PLA(2) and thus provides new threads for the study of the functions and structures of snake venom PLA(2)s. One phosphorylation site and the size of the glycan chain are determined by using HPLC/nESI-MS/MS and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. The delicate utilization of ESI-MS can exert tremendous impact on protein sciences.
高效液相色谱/纳米电喷雾电离串联质谱（HPLC/nESI-MS/MS）结合二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（2D SDS-PAGE）的结果表明，磷脂酶A(2)（PLA(2)）的单体和二聚体共存于中国东北白眉蝮蛇粗毒（ABUSV）中。然后，从ABUSV中纯化出一种精确分子量为13979.6 Da的酸性PLA(2)（mo-ABUSV-aPLA(2)）。将ABUSV-aPLA(2)的串联质谱衍生肽段与其他同源蛇毒PLA(2)进行比较，结果表明ABUSV-aPLA(2)是一种新型蛇毒PLA(2)。同时，还获得了ABUSV-aPLA(2)二聚体（di-ABUSV-aPLA(2)）。串联质谱分析鉴定出di-ABUSV-aPLA(2)与mo-ABUSV-aPLA(2)具有相同的肽段，这表明di-ABUSV-aPLA(2)是同源二聚体。每个ABUSV-aPLA(2)含有一个Ca(2+)离子。该Ca(2+)离子对ABUSV-aPLA(2)的水解活性和结构都至关重要。Pro-Q Emerald和Pro-Q Diamond特异性糖蛋白和磷蛋白染色结合串联质谱分析表明，ABUSV-aPLA(2)既是一种糖蛋白也是一种磷蛋白，据我们所知，这是关于蛇毒PLA(2)的首次此类报道，从而为蛇毒PLA(2)的功能和结构研究提供了新线索。通过使用HPLC/nESI-MS/MS和基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱确定了一个磷酸化位点和聚糖链的大小。电喷雾电离质谱的巧妙应用可对蛋白质科学产生巨大影响。
