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', approximately 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', approximately 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 4-9 kDa 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-Gly(27)-pilosulin 2, approximately 3155 Da) and a previously unreported peptide of approximately 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 approximately 6067 Da variant, in which the valine at residue 5 is replaced by isoleucine. A range of hydrolysis products of [Ile(5)]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.