Venom Proteome of the Box Jellyfish Chironex fleckeri
Total protein from CFV in a Coomassie-stained 15% SDS-PAGE gel. Lanes were divided into 40 gel slices (dotted lines) and subjected to in-gel tryptic digest before LC-MS/MS analysis. Markers (M) are indicated and the numbering system used for gel slices. CFV proteins separated using SDS-PAGE and stained in-gel with fluorescent dyes reactive to glycans or phosphate groups (Lanes 1–4). Glycan analysis showed fluorescence in bands corresponding to CfTX proteins (Lane 1) and no fluorescence in the negative control (Lane 2). No phosphorylation was observed except in band 40 (Lane 4; positive control in Lane 3). Western blot analysis using polyclonal antibodies for CfTX-1 and -2 showed hybridisation in two bands corresponding to the highest scoring Mascot identifications for these proteins and in the region corresponding to approximately 12 kDa (lane 5). No other bands were positive for these proteins despite their identification in MS/MS analysis. The spectral counts for proteins from this toxin family identified using Mascot are displayed adjacent to the band numbering (blue, red and green lines for CfTX-1, CfTX-2 and CqTX-A resp.). Actual spectral counts for a selection of CfTX-1 points are shown for reference.