Proteomic Identification of Marine Protein Toxin

Introduction: Stonefish have caused a number of human deaths, but the protein profiles of the venom have not been clearly established due to the venom's extreme lability.

Purpose: Performace of feasibility of proteomic approach in identifying fish spine venom.

Methods: To elucidate the toxin components, native polyacrylamide gel electrophoresis (Native-PAGE), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) were employed in this study to establish the protein pattern profiles of crude venom from Synanceia verrucosa in Taiwan. After in-gel digestion, a matrix assisted laser desorption/ionization-quadrupole time of flight mass spectrometer (MALDI-Q-TOF) was used to identify protein samples. MALDI-Q-TOF can analyze PMF and further provide product ion spectra (MS/MS ion search) for protein identification.

Results: The venom estimated by Native-PAGE were 471, 358, 260, and 166 kDa. The predominate protein bands of crude venom were 84 and 75 kDa by SDS-PAGE. The crude venom protein fell in the region with pI values of 7 to 9 and molecular weights of 75 to 90 kDa by 2-DE. Peptide mass fingerprints (PMF) and MS/MS ions originated from MALDI-Q-TOF were used to identify the protein. Our results showed that the complete components of neoverrucotoxin (neoVTX) α-subunit and β-subunit as well as verrucotoxin (VTX) β-subunit were identified from SDS-PAGE and 2-DE patterns.

Significance: This study was to demonstrate the efficacy of current bottom-up proteomic and bioinformatic approaches in identifying proteinaceous venom toxins from S. verrucosa spines.