著者

Shimba Kawasue Yohei Sakaguchi Reiko Koga Tadashi Hayama Hideyuki Yoshida Hitoshi Nohta

出版者

The Pharmaceutical Society of Japan

雑誌

Chemical and Pharmaceutical Bulletin (ISSN:00092363)

巻号頁・発行日

vol.70, no.1, pp.19-24, 2022-01-01 (Released:2022-01-01)

参考文献数

30

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Casein is one of the allergen proteins present in milk. Therefore, a quantification method for the selective analysis of casein using fluorous derivatization with LC-tandem mass spectrometry (LC-MS/MS) was developed. After two allergen proteins (αS1-casein and β-casein) extracted from baked sugar cookies were tryptic digested, the obtained phosphorylated peptides were selectively derivatized by β-elimination with Ba(NO3)2 under basic condition and Michael addition with perfluoroalkylthiol (1H,1H,2H,2H-perfluorooctanethiol, PFOT). In this study, YKVPQLEIVPN(pSer)AQQR (104–119 fragment from αS1-casein) and FQ(pSer)EEQQQTEDELQDK (33–48 fragment from β-casein) obtained by tryptic digestion were selected as target peptides. The phosphorylated serine residue in each peptide was converted to a perfluoroalkyl group by derivatization. The obtained fluorous-derivatized peptides were analyzed by LC-MS/MS, to which a fluorous LC column was connected. Therefore, it was possible to analyze casein without being affected by the matrix components in the baked food sample. When the present method was applied to cookies with arbitrary amounts of αS1-casein and β-casein, the obtained quantification values were in good agreement with the arbitrary amounts spiked. The quantification limits of αS1- and β-casein in cookie analysis were 246 and 152 ng/g, respectively. Hence, this method can be used to analyze trace amounts of allergen proteins present in the baked food.

著者

Ryoko TOMITA Kenichiro TODOROKI Hiroshi MARUOKA Hideyuki YOSHIDA Toshihiro FUJIOKA Manabu NAKASHIMA Masatoshi YAMAGUCHI Hitoshi NOHTA

出版者

The Japan Society for Analytical Chemistry

雑誌

Analytical Sciences (ISSN:09106340)

巻号頁・発行日

vol.32, no.8, pp.893-900, 2016-08-10 (Released:2016-08-10)

参考文献数

22

被引用文献数

1 12

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We performed a comprehensive quantification of 20 amino acids in RPMI 1640 medium-cultured human colorectal adenocarcinoma cells to evaluate the efficacy of 5-fluorouracil treatment under hypoxic and hypoglycemic conditions, which mimic the tumor microenvironment. In this study, we developed a simple and comprehensive analytical method by using LC-MS/MS connected to the Intrada amino acid column, which eluted amino acids within 9 min. The present method covered a linearity range of 3.6 – 1818 μM, except for Gly (227 – 1818 μM), Ala, Asp, His (7.1 – 1818 μM each), and Trp (3.6 – 909 μM). The limits of detection were in the range of 0.02 – 38.0 pmol per injection in a standard solution. Amino acid concentration data were analyzed using principal-component analysis to represent samples on two-dimensional graphs. Linear discriminant analysis was used to classify samples on the score plots. Using this approach, the effect of 5-fluorouracil treatment could be successfully discriminated at high discrimination rates. Moreover, several amino acids were extracted from corresponding loading plots as candidate markers for distinguishing the effects of the 5-fluorouracil treatment or tumor microenvironmental conditions. These results suggest that our proposed method might be a useful tool for evaluating the efficacy of anticancer drugs in the tumor microenvironment.