Characterisation of major peptides in "Jack Jumper" ant venom by mass spectrometry

The jack jumper ant, Myrmecia pilosula, is endemic to South-Eastern Australia, where around 2.7% of the population has a history of systemic allergic reactions (anaphylaxis) to its venom. Previous work had indicated that there were several allergenic peptides derived from the cDNA Myr p 1, the major expressed allergenic product being a 56-residue peptide (Myr p 1 57->112, "pilosulin 1", ~6052 Da). Another major allergen had been described as a 27 residue peptide derived from the cDNA Myr p 2 (Myr p 2 49->75, "pilosulin 2", ~3212 Da), possibly existing as part of a disulfide complex. As a preliminary step in detailed stability studies of a pharmaceutical product used for venom immunotherapy, LC-MS and Edman sequencing analysis of venom collected from various locations by both electrical stimulation and venom sac dissection was undertaken. More than 50 peptides in the 4kDa to 9kDa range were detected in LC-MS analyses. A subsequence of Myr p 2 was found as part of the major peptide present in all samples; this was a bis-disulphide linked, antiparallel aligned heterodimer consisting of Myr p 2 49->74, (des-Gly27-pilosulin 2, ~3155 Da) and a previously unreported peptide of ~2457 Da. Pilosulin 1 was found by a combination of tandem mass spectrometry and Edman sequencing to exist mainly, and sometimes exclusively, as a previously unreported ~6067 Da variant, in which the valine at residue 5 is replaced by isoleucine. A range of hydrolysis products of [Ile5]pilosulin 1 and pilosulin 1 were also detected in partially degraded venom. Further IgE-binding studies using these peptides are warranted and a revision of the nomenclature of allergenic components of M. pilosula venom may be required to conform with established IUIS guidelines