Analysis of Allergenic Proteins by Mass Spectrometry

The general aim of the activities conducted in the framework of my PhD were to learn modern techniques for analyzing proteins by mass spectrometry (MS) and then to apply these techniques and approaches to the analysis of allergenic proteins contained in foods. My research activities were conducted at the Laboratory of Protein Chemistry of CRIBI, University of Padua, where previously I have conducted the research for my Thesis for the Doctor degree in Pharmaceutical Biotechnologies. During the fist year of my PhD I have concluded the Thesis project on the amyloid aggregation of -lactalbumin, a model protein utilized for investigating molecular aspects of protein amyloidogenesis. The results of this research were quite interesting and indeed they have been published in an international journal. During the first two years I acquired a solid knowledge on several aspects of the MS methodology and I was able to learn the theory and practice of several modern techniques and approaches in this ambit. The specific aim was to analyze allergenic proteins contained in complex matrices as foods and to this aim several proteins were extracted and purified from several food samples. The research has been focused mostly on the allergenic proteins from milk and eggs, known to cause widespread allergies. The proteins of interest were analyzed by using several chromatographic and electrophoretic techniques and also by means of HPLC connected to a tandem MS electrospray instrument. I was able to show that MS techniques can be used to identify allergenic proteins even when contained in very complex mixtures. Therefore, these MS techniques perhaps can be used as an alternative to the immunochemical methods nowadays in use for detecting allergens. I have also analyzed the chemical modifications that allergenic proteins suffer during several industrial treatments of foods, including heat treatment. During the third year of my PhD I spent a six months period at the Biochemistry Laboratory of the Imperial College in London, being involved in a project aimed to study in a large scale the proteins of the mosquito Anopheles gambiae. The MS analyses were focused on the proteins responsible of the mating behaviour of A. gambiae, hoping to identify a target for controlling the behaviour of this vector of the malaria disease. Summing up, besides the publication dealing with amyloid aggregates of beta- lactalbumin, this PhD Thesis is composed by a major part dealing with MS analysis of allergenic proteins and by a minor one dealing with MS analysis of proteins from A. gambiae.