In-Gel Digest to identify Aspergillus fumigatus protein antigens in severe equine asthma (SEA)

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.