Aspergillus fumigatus (Asp f) has been described as a major extrinsic stimulus of SEA and is a common mold species in hay. Aiming to identify disease-relevant antigens, we analyzed Asp f as a source, in an immuno-proteomics approach on two-dimensional immunoblots. For 21 of 289 spots serum Ig binding was different between asthmatic and healthy horses’ sera. Proteins were in-gel digested and tryptic peptides extracted from the 21 spots of interest, analyzed by liquid chromatography-mass spectrometry and eight identified and prioritized candidate antigens were expressed as recombinant proteins.

Peptides obtained after tryptic in-gel digest were analyzed with a nanoACQUITY Ultra Performance LC™ coupled to a Q-TOF SYNAPT G2-Si instrument. For measurments a high-definition data-dependent acquisition approach (HD-DDA) for top 6 ions was used. Followed by data analysis with Mascot Distiller (Version 2.8.1.0) and Mascot search engine (Version 2.7.0).

66 in-gel digest samples from 2D-PAGE spots for identification of protein antigens; 19 in-gel digest samples from SDS-PAGE bands to verify protein expression