Key words: multidimensional liquid chromatography (MDLC) • reversed-phase chromatography (RPC) • LC-MS/MS • DeCyder MS • differential analysis
Ettan™ MDLC was used in conjunction with tandem mass spectrometry (MS/MS) to separate and identify allergenic proteins extracted from the pollen of the olive tree, Olea europaea. DeCyder™ MS Differential Analysis Software (DeCyder MS) was used to quantitate differentially expressed proteins in two different samples, Ole_e1_"mer" and Ole_e1_"lite."
Ole e 1 is the most frequent sensitizing agent affecting more than 70% of the patients suffering from olive (Olea europaea) pollinosis. The protein consists of a single polypeptide chain of 145 amino acids (MW 16.3 kDa) and has been suggested to be involved in the hydration of pollen during germination (1).
Ole e 1 exists in several isoforms, displaying microheterogeneities, that are difficult to resolve by gel-based methods. Therefore, we used an LC-MS/MS protein separation and identification strategy.
DeCyder MS Differential Analysis Software (DeCyder MS) is a tool for visualization, detection, comparison, and label-free relative quantitation of LC-MS/MS data (2). In brief, the software displays LC-MS analyses as two-dimensional intensity maps, resulting in a much better overview of spectral quality and data compared with the traditional way to present LC-MS data. Statistical analysis is carried out on all peptide spots within minutes.
The DeCyder MS algorithm is based on accurate mass and reproducible retention times. Ettan MDLC is ideally suited to deliver reproducible retention times (3), and should be used to generate the data that will be analyzed by DeCyder MS.
In this application note, relative quantitation of allergenic proteins in two