As expected, such annotation revealed many transcripts related to molecules involved in either primary metabolic pathways ((short ragwed) causes severe allergic rhinoconjunctivitis [8]. To circumvent this problem, we applied a broad RNA sequencing approach to first generate a comprehensive inventory of the short ragweed pollen transcripts, then infer proteins and further characterize the proteome by mass spectrometry (MS) [12C15]. Combining this approach with IgE reactivity profiling of individuals sera using high-resolution 2D-gel electrophoresis, we present herein a detailed characterization of the pollen proteome and allergome, with evidence for a number of allergens and isoallergens. Methods Analysis and annotation of the short ragweed pollen transcriptome Total RNAs were isolated from pollen grains (GREER, Lenoir, NC) using the RNeasy kit (Qiagen, Courtaboeuf, France). The selection of mRNAs and the construction of a random primed library were carried out by Vertis Biotechnologies (Freising, Germany). Subsequent deep mRNA sequencing TM5441 using a 454 sequencing apparatus with titanium chemistry (Roche Diagnostics, Meylan, France), assembly with the Newbler software (Roche Diagnostics) and annotation were performed by Beckman Coulter Genomics (Grenoble, France). For each transcript, 6-framework translations were used to perform a BLASTP analysis against proteins available for the flowering vegetation (taxonomy ID 3398) and determine probably the most probable reading frames. An annotated transcriptome-derived proteome TM5441 (TDP) database containing 9678 expected protein sequences was built based upon translation(s) in framework(s) yielding a blast hit, or providing the longest translated sequences. Protein sequences shorter than 33 amino acids and without any blast hit TM5441 (e-value above 10?5) were excluded. To identify putative short ragweed allergens by similarity to known allergens, the TPD entries were compared by BLASTP to a set of plant proteins labeled as allergens in Uniprot (and allergen) using the CLC Genomics Workbench 7 software (CLCbio, Aarhus, Denmark). A hit was considered as positive when the determined e-value was smaller than 10-5. A pathway analysis was performed within the put together transcripts with the KEGG (Kyoto Encyclopedia of Genes and Genomes) Automatic Annotation Server (KAAS). KEGG orthologies (KO) were assigned using the bidirectional best hit (BBH) method and as a gene data arranged. Analysis of the short ragweed pollen proteome Short ragweed pollen grains were floor in liquid nitrogen, then resuspended at 1:5 (w/v) in PBS pH 7.4 (Ambion, Austin, TX) supplemented having a cocktail of protease inhibitors (Complete, Roche, Meylan, France). After mild shaking at space temp for 1 hour and centrifugation at 10,000 for 30 min, supernatants were collected, filtered at 0.22 m, FLT3 and enriched in low large quantity proteins using the Proteominer kit (Bio-Rad, Marne La Coquette, France). Proteins were digested with trypsin prior to analysis by reversed-phase liquid chromatography using an Ultimate 3000 RS nano LC system (Thermo Fisher Scientific, Villebon sur Yvette, France) coupled to MS (Effect HD, Bruker Daltonics, Wissembourg, France). Peptide recognition was performed using the PEAKS software (Bioinformatics Solutions Inc., Waterloo, Canada) and the in-house TDP database supplemented with Amb a 4, Amb a 5, Amb a 6 sequences from IUIS (www.allergen.org) as they were not retrieved through our transcriptome analysis. Only proteins recognized with a minimum of 2 peptide sequences, including at least 1 unique sequence, were taken into account. Further details on MS analysis and protein identification are provided in the online repository (In-solution MS analyses). To create a proteome map, proteins from an aqueous short ragweed pollen draw out were first precipitated using the PerfectFocus kit (Agro-Bio, La Fert Saint Aubin, France), resuspended inside a 7 M urea, 2 M thiourea, 4% CHAPS and 30 mM Tris pH 8.8 buffer, before 2D-gel electrophoresis using 3C10 non linear pH range 12.5% DALT gels (GE Healthcare, Velizy-Villacoublay, France), as per the manufacturers instructions. Following Sypro Ruby staining (Existence Systems, Saint Aubin, France), protein spots of interest were excised from 2D-gels using an EXquest spot cutter (Bio-Rad), then submitted to tryptic digestion and analyzed by LC-MS/MS. Further details on MS analyses and protein identification are provided in the online repository (In-gel MS analyses). Recognition of IgE reactive proteins in short ragweed pollen Sera from 22 Western patients sensitive to short ragweed pollen, enrolled in a phase I study (ClinicalTrials.gov identifier: “type”:”clinical-trial”,”attrs”:”text”:”NCT01224834″,”term_id”:”NCT01224834″NCT01224834) after authorization by a local ethical committee, Medical Study Council Ethics Committee for Clinical Pharmacology (Hungary; EudraCT: 2008-003715-12), were used to detect short ragweed pollen proteins exhibiting IgE reactivity. Written educated consents were from patients. All individuals had a.
CYP