Using Stable Isotope Dimethyl Labeling Coupled with Mass Spectrometry to Detect Tomato Allergen Sola l 1

　　In Taiwan, tomato is one of the most important vegetables that frequent causes of allergy. Recently, Sola l 1 was confirmed as a new minor allergen in tomato fruits. Because of the highly conserved sequence of protein structure among profilins, highly cross-reactive usually happen between pollen and foods and then caused allergic symptoms. Immunization assay is the most common method to detect food allergens. However, it may lead to false positive or false negative results due to the presence of cross-reactions. Therefore, we must to seek a specific method to analyze tomato allergens. Mass spectrometry has the advantage of high specificity, high sensitivity, high accuracy and rapid analysis, so it is used in many different fields for qualitative and quantitative analysis. Multiple reaction monitoring (MRM) is the most common method for quantitation with tandem mass spectrometry. Stable isotope dimethyl labeling has been widely used in quantitative proteomics due to it is highly efficient, fast, simple and inexpensive, but nobody applies it to vegetable protein sample. The purpose of this study was to develop a novel strategy by using stable isotope dimethyl labeling with LC - MS/MS, and establish the analysis platform for tomato allergen Sola l 1 then applied to the fields of food analysis. 　　First, we analyzed the recombinant protein rSola l 1 by trypsin digestion and identified its sequence by Q-TOF, then found a suitable peptide as the standard peptide of target protein. The qualitative result showed that the peptide YR, which has 13 amino acids in length, was stable and unique. The standard peptide was synthesized and used to synthesized for further experiments. Second, the synthetic peptide YR was labeled with formaldehyde-H2 and formaldehyde-D2, respectively. The dimethylated tryptic peptides were subsequently analyzed by Q-TOF. The result indicated the YR peptides obtain a complete labeling of N-terminal residue, and it represented that the method equipped with a good labeling efficiency. A series of different concentration of light stable isotope dimethyl-labeled synthetic peptides (YR-H) were prepared, and fixed concentration of intermediate stable isotope dimethyl-labeled synthetic peptides (YR-D) were spiked as internal standards. The calibration curve was obtained and indicated a good linear relationship of the concentration of YR-H between 0.5 ng/mL to 2500 ng/mL, and it could be used for quantitation of target protein. Further, we validated the method through evaluating its linearity, recovery, limit of detection and quantification and repeatability, the result showed that dimethyl-labeled could be applied to recombinant protein and standard peptides, and it was reliable and accurate. The detection and quantification limits (LOD and LOQ) that analyzed by MRM of recombinant protein were both 0.5 μg/mL, and standard peptides were 0.1 ng/mL and 0.5 ng/mL, respectively, and this method showed potential for comprehensive quantification of protein.