Electrospray ionization mass spectrometry as a critical tool for revealing new properties of snake venom phospholipase A2.
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.
Liu
Shuqing
S
Department of Biochemistry & Molecular Biology, Dalian Medical University, Dalian, 116044, China.
Zhang
Cuili
C
Xu
Yue-Fei
YF
Yang
Fan
F
Sun
Ming-Zhong
MZ
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000737:chemistry* / Q000201:enzymology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D054467:Phospholipases A2 / Q000737:chemistry* / Q000302:isolation & purification; D010766:Phosphorylation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D003435:Crotalid Venoms; D054467:Phospholipases A2