Electrospray ionization mass spectrometry as a critical tool for reveallng new properties of snake venom phospholipase A2

Abstract

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 A2 (PLA 2) coexisted in crude Chinese Agkistrodon blomhoffii Ussurensis snake venom (ABUSV). Then, an acidic PLA2 with the accurate molecular mass of 13979.6 Da was purified from ABUSV (mo-ABUSV-aPLA2). MS/MS-derived peptides from ABUSV-aPLA2 were compared with other homologous snake venom PLA2s, which in turn showed that ABUSV-aPLA2 is a novel snake venom PLA2. Meanwhile, the ABUSV-aPLA2 dimer (di-ABUSV-aPLA2) was also obtained. MS/MS analysis identified the same peptides from di-ABUSV-aPLA2 as from mo-ABUSV-aPLA2, which indicates that di-ABUSV-aPLA2 is a homodimer. One Ca2+ ion is contained per ABUSV-aPLA2. The Ca2+ ion is critical for both the hydrolytic activity and the structure of ABUSV-aPLA2. Pro-Q Emerald and Pro-Q Diamond specific glycoprotein and phosphoprotein staining combined with MS/MS analysis indicated that the ABUSV-aPLA2 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 PLA2s. 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. Copyright © 2009 John Wiley and Sons, Ltd.