Analysis of allergenic proteins in sweet cherry by liquid chromatography/tandem mass spectrometry using stable isotope-labeled peptides

Pru av 1, Pru av 3, and Pru av 4 are main allergenic proteins present in sweet cherry (Prunus avium L.). In this study, an analytical method was developed for these proteins based on a protein absolute quantification (AQUA) strategy. This method involves performing liquid chromatography/tandem mass spectrometry (LC/MS/MS) using stable isotope-labeled internal standard (SIIS) peptides, LVASPSGGSIIK[13C6, 15N2], ISPSTNC[CAM]ATVK[13C6, 15N2] (C[CAM]: carbamidomethyl-modified C), and LGDYLIEQGL[13C6, 15N], for the analysis of Pru av 1, Pru av 3, and Pru av 4, respectively. This method showed linear relationships (r2 > 0.99) in concentration ranges of 0.4–200, 0.2–200, and 1.6–200 fmol/μL of LVASPSGGSIIK[13C6, 15N2], ISPSTNC[CAM]ATVK[13C6, 15N2], and LGDYLIEQGL[13C6, 15N], respectively, spiked into the matrix of a tryptic digest from sweet cherry. However, the overall coefficients of variation for inter-day tests were unsatisfactory. Pru av 1, Pru av 3, and Pru av 4 present in four cultivars of sweet cherry could be detected using this method. This method is expected to detect and semi-quantify allergenic proteins in sweet cherry fruits as an alternative to western blotting.