In-band identification by MaxQuant software for allergen discovery

The experiment workflow to identify the immunoreactive food allergens is well established. After extraction of allergens from the food matrix, the proteins are separated by electrophoretic technique, blotted on nitrocellulose membranes and immunorecognised by the allergic patient's IgEs. The immunereactive bands are cut, digested by trypsin and identified by MS technique. In our set-up, after LC-MS/MS analysis, protein identification is performed by using MaxQuant software. In order to discover which is the "allergen" among all the proteins present in the band, we have to: i) identify all the band-contained proteins, ii) evaluate their relative abundance and iii) find out which is the protein involved in patient's IgE binding. Considering that MaxQuant default setting is designed for off-gel comparative proteomics, to extend its applicability to our purposes we modified some parameters (i.e. peptide to be included in the identification, the score value of modified and unmodified peptides, iBAQ calculation). The comparison between default and our setting will be presented.