Novel IgE Recognized Components of Lolium perenne Pollen Extract: Comparative Proteomics Evaluation of Allergic Patients Sensitization Profiles Michele De Canio, †,# Simona D’Aguanno, ‡,# Cristiano Sacchetti, † Francesca Petrucci, § Giovanni Cavagni, | Marzia Nuccetelli, ⊥ Giorgio Federici, †,| Andrea Urbani,* ,†,‡,O and Sergio Bernardini †,|,O Department of Internal Medicine, University of Rome Tor Vergata, Rome, Italy, Laboratory of Proteomics and Metabonomics, S. Lucia Foundation-IRCCS, Rome, Italy, Department of Biomedical Science, University “G. D’Annunzio”, Chieti, Italy, Children’s Hospital “Bambino Gesu ` ”-IRCCS, Rome, Italy, and Department of Laboratory Medicine, Policlinico Tor Vergata Foundation, Rome, Italy Received April 6, 2009 Abstract: In the last years, proteomic investigation pro- vided a powerful tool in molecular characterization of complex allergen sources with relevant implications in both diagnosis and immunotherapic treatment of aller- gies. We followed a proteomic approach to characterize ryegrass (Lolium perenne) pollen, a common cause of seasonal allergic diseases affecting an increasing part of world population. Peptide shotgun experiments per- formed on nanoLiquid Ultra Pressure Chromatography coupled with fast Q-TOF MS-MS/MS acquisition protocols (MS E ) and 2-DE immunoblot combined with MALDI-TOF- TOF analysis allowed the detection of all previously identified ryegrass allergens. Comparative analysis of immunoblot highlighted a class of patients characterized by a more complex 2-DE pattern associated with in- creased levels of IgE antibodies and by higher susceptibil- ity to multiple sensitization toward different allergen sources. Cluster analysis revealed that all these patients recognized profilin, considered the main cross-reactive allergen in grass pollen. Furthermore, mass spectrometry analysis revealed the presence of other IgE reactive components in ryegrass pollen that might be involved in polysensitization, such as cyclophilin, fructosyltransferase and legumin-like protein. Keywords: IgE binding protein • pollen allergens • mass spectrometry • LC-MS/MS 1. Introduction Allergic diseases are Immunoglobulin E (IgE)-mediated type I hypersensitivity reactions against common environmental substances. Atopic individuals respond to allergens exposure by production of IgE antibodies, leading basophils and tissue mast cells to secrete immuno and inflammatory response mediators, such as histamine, cytokines, leukotrienes. The final effect is the elicitation of allergic symptoms, ranging from dermatitis or rhinoconjunctivitis to bronchial asthma. 1 Pollens are the most frequent cause of seasonal allergic rhinitis (SAR) affecting about 15% of European population. 2 In literature, allergic reactions have been described against pollens from a variety of plant species. Only the Poaceae family (grasses) contains about 25% of allergenic pollen species and about 45% of sequenced allergens. 3 Grass pollen allergens may have elevated sequence homology and exhibit similar biochemical and im- munological properties. 4 According to these features, the Inter- national Union of Immunological Societies (IUIS)-Allergen No- menclature Sub-Committee (www.allergen.org) classified grass pollen allergens into 11 groups. However, molecular variability due to allelic polymorphism, post-translational modifications and alternative mRNA splicing can be also observed among allergens from the same group. Groups 1 and 5 include major allergens, recognized by IgE of about 95% and up to 85% of grass pollen- sensitized patients, respectively. Group 2/3 is recognized with a frequency of approximately 60%. 5 In Lolium perenne (ryegrass), six allergen groups are re- ported. Lol p 1 (group 1) is a glycoprotein of 31-35 kDa, belonging to the -expansins family of papain-related cysteine proteinases, involved in the loosening of plant cell walls during the extension. 6 Lol p 2 and Lol p 3 are 11-12 kDa related proteins, showing sequence similarity to the C-terminal do- mains of group 1 allergens. 7 Lol p 4, a basic 50-67 kDa protein, containing a FAD binding domain, 8,9 and Lol p 11, a soybean trypsin inhibitor-related protein, 10 are glycosilated allergens. Lol p 5 isoallergens consist of two main isoforms, A and B, showing 66% sequence identity 11 with an apparent molecular mass of 27-33 kDa. Finally, cytochrome C was characterized as allergen and named Lol p 10, 12 but further investigations have not supported its relevancy. 4 * To whom correspondence should be addressed. Prof. Andrea Urbani, University of Rome “Tor Vergata”, Dept. of Internal Medicine, Via Montpellier 1, 00133 Rome, Italy. E-mail: andrea.urbani@uniroma2.it. † University of Rome Tor Vergata. ‡ S. Lucia Foundation-IRCCS. # These authors made equal contributions and should be considered as “first authors”. § University “G. D’Annunzio”. | Children’s Hospital “Bambino Gesu ` ”-IRCCS. ⊥ Policlinico Tor Vergata Foundation. O Both authors acted as senior investigators and should be considered equal “last authors”. 10.1021/pr900315a CCC: $40.75  2009 American Chemical Society Journal of Proteome Research 2009, 8, 4383–4391 4383 Published on Web 07/08/2009