Protein samples were obtained from the pollen of the olive tree, Olea europaea, by aqueous extraction at low pH. The two samples used in this study, Ole_e1_"mer" and Ole_e1_"lite," refer to different batches of collected pollen containing varying amounts of the allergen.
Two mg each of Ole_e1_"mer" and Ole_e1_"lite" was partially purified using an Ettan LC System. The chromatographic separation was performed on a Superdex 75 10/300 GL gel filtration column at 0.5 ml/min in 80 mM sodium acetate, pH 4.5, 50 mM NaCl, and 0.5-ml fractions were collected using Fraction Collector Frac-950. Fractions were examined using MALDI-ToF MS. Those containing proteins with molecular weights 14–20 kDa were pooled and dried in vacuo.
Protein reduction, alkylation, and trypsin digestion
The pooled, dried fractions from the gel filtration step were dissolved in 0.5 ml of 9 M urea containing 50 mM DTT and incubated at ambient temperature for 60 min. An equal volume of a solution consisting of 8 M urea, 250 mM Tris-HCl, pH 8.8, and 125 mM iodoacetamide was added, and incubation was continued for another 60 min in darkness. The samples were desalted against 20 mM ammonium bicarbonate on NAP-10 Columns, and then digested with 5 µg trypsin for 2 h at 37 °C. The digestions were stopped by acidification with formic acid.
Dual reversed-phase nanoscale liquid chromatography
Ettan MDLC was used in the high-throughput configuration using two reversed-phase (RPC) trap columns 300 µm i.d. x 5 mm (Zorbax 300-SB C18) for online desalting and sample cleanup, followed by two nanoscale RPC analytical columns 75 µm i.d. x 150 mm (Zorbax 300-SB C18) for high-resolution separation. One set of RPC trap/analytical columns was equilibrated while the second set