The graph extracted from the PepMatch module in Figure 6 shows the sequences and signal intensities of the significantly varying peptides that were used for quantitation of different isoforms of Ole e 1. As earlier mentioned, LGMYPPNM is present in all forms of Ole e 1, resulting in the highest signal intensity or most blackened grayscale in the intensity maps created by DeCyder MS. The other peptide pairs in Figure 6 thus represent unique peptides for five different isoforms of the olive allergen.
In Table 1, a list of significantly varying peptides from the two allergen samples is shown, together with Student t-test p values, protein names, pI values, molecular masses, and accession numbers. As can be seen, several of the isoforms were very close in molecular mass and pI value. The smallest deviation in mass was 64 Da between Ole 1 c (16485 Da) and Ole e 1.0103 (16421 Da), while the smallest deviation in pI value was 0.02 units between Ole e 1 (CAA73036.1) and Ole e 1.0103 (CAA73037.1).
DeCyder MS Differential Analysis Software is a tool for the visualization and label-free relative quantitation of complex protein digests. The precise gradient formation and solvent delivery features of Ettan MDLC provided highly reproducible retention times for eluted peptides, resulting in improved matching performance by DeCyder MS.
Quantitation of allergen samples using DeCyder MS revealed a 3.8-fold difference in Ole e 1 content, which is in agreement with other physicochemical quantitation methods. In addition, five differentially regulated isoforms of Ole e 1 could be unambiguously identified and quantitated.
The results indicate that for quantitation of proteins containing microheterogeneities, the LC-MS based proteomics workflow combined with DeCyder MS evaluation could be preferable to the gel-