Fractions A4–A6 were pooled because they contained proteins varying in size from 14 to 20 kDa, thus covering the expected molecular weight of Ole e 1 according to analyses by MALDI-ToF MS (data not shown). As can be seen from the chromatograms, Ole_e1_"mer" contained about 3.5 times more material in the collected fractions in comparison with Ole_e1_"lite," as judged by integration of the UV traces of the collected fractions.
Nanoscale LC-MS/MS of tryptic allergen peptides
Fractions A4–A6 from the gel filtration analyses of Ole_e1_"mer" and Ole_e1_"lite," respectively, were pooled, digested with trypsin, and analyzed by LC-MS/MS using the Ettan MDLC coupled to a Finnigan LTQ linear ion trap mass spectrometer. The resulting base peak ion chromatogram of Ole_e1_"lite" is shown in Figure 2.
The full scan MS data was acquired in profile mode, which is required in order to get enough data points for evaluation by DeCyder MS, while MS/MS data (not used for matching) was collected in centroid mode. The other batch, Ole_e1_"mer," displayed the same base peak chromatogram profile (data not shown). The generated MS and MS/MS files were transferred to TurboSequest software, which resulted in identification of about 40 proteins with at least two peptide hits for each protein from each of the examined samples. For each batch the highest ranked protein candidate was identified as the main olive allergen.
Detection of olive allergens using DeCyder MS
The individual intensity maps created from the Ettan MDLC-MS/MS analyzed fractions of Ole_e1_"mer" and Ole_e1_"lite" were examined