Nagai Hiroyuki, Minatani Tomiaki, Goto Kotaro
Gifu Prefectural Research Institute for Health and Environmental Science, 1-1 Naka-Fudougaoka, Kakamigahara, 504-0838, Japan.
J AOAC Int. 2015 Sep-Oct;98(5):1355-65. doi: 10.5740/jaoacint.14-248.
An LC/MS/MS analysis method was developed for crustacean allergens, tropomyosin, and arginine kinase. A protein extract from shrimp was reduced, alkylated, and digested by trypsin. Peptide spectra were obtained using full scan analysis by LC/MS/MS, and we determined a sequence through a protein search. 22ADTLEQQNK30, 92IQLLEEDLER101, 113LAEASQAADESER125, 134SLSDEER140, 153FLAEEADR160, and 190IVELEEELR198 of tropomyosin and 152VSSTLSSLEGELK164 and 217TFLVWVNEEDHLR229 of arginine kinase were selected as the specific peptides, and optimal multiple-reaction monitoring conditions were used. The results obtained through the LC/MS/MS analysis correlated well with those using the ELISA method for various crustacean samples (r2>0.9). Moreover, unregulated species, such as krill or insects, which produce positive results in some crustacean ELISA assays, can be differentiated by LC/MS/MS. These findings suggest that LC/MS/MS analysis may be effective for crustacean food allergen analysis.
开发了一种用于分析甲壳类过敏原、原肌球蛋白和精氨酸激酶的液相色谱串联质谱（LC/MS/MS）分析方法。虾的蛋白质提取物经过还原、烷基化处理，并用胰蛋白酶消化。通过LC/MS/MS全扫描分析获得肽谱，并通过蛋白质搜索确定序列。选择原肌球蛋白的22ADTLEQQNK30、92IQLLEEDLER101、113LAEASQAADESER125、134SLSDEER140、153FLAEEADR160和190IVELEEELR198以及精氨酸激酶的152VSSTLSSLEGELK164和217TFLVWVNEEDHLR229作为特异性肽段，并使用了最佳的多反应监测条件。通过LC/MS/MS分析获得的结果与使用酶联免疫吸附测定（ELISA）方法对各种甲壳类样品获得的结果相关性良好（r2>0.9）。此外，在某些甲壳类ELISA检测中呈阳性结果的未规范物种，如磷虾或昆虫，可以通过LC/MS/MS进行区分。这些发现表明，LC/MS/MS分析可能对甲壳类食品过敏原分析有效。
