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Gelatin Quantification by Oxygen-18 Labeling and Liquid Chromatography–High-Resolution Mass Spectrometry

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journal contribution
posted on 10.12.2014 by Xiao-Mei Sha, Zong-Cai Tu, Hui Wang, Tao Huang, Deng-Le Duan, Na He, De-Jun Li, Hui Xiao
Combined with high-performance liquid chromatography (HPLC) and linear-ion trap/Orbitrap high-resolution mass spectrometry, trypsin-catalyzed 16O-to-18O exchange was used to establish an accurate quantitative method for bovine or porcine gelatin. The sophisticated modifications for these two mammalian gelatins were unambiguously identified by accurate mass and tandem mass spectrometry. Eighteen marker peptides were successfully identified for the bovine and porcine gelatin, respectively. The gelatins were subjected to 18O or 16O labeling in the presence of trypsin and mixed together in various ratios for quantification. All of the 18O-labeled peptides were also confirmed by accurate mass and tandem mass spectrometry. The 10 marker peptides with the strongest signals were chosen to calculate the average ratios of 18O-labeled and 16O-labeled gelatin. The measured ratios of 18O-labeled and 16O-labeled peptides were very close to the mixing ratios of 20:1, 5:1, 1:1, and 1:5 with low standard deviation values. The samples with a mixing ratio of 1:1 18O-labeled and 16O-labeled peptides were determined to 1.00 and 0.99 with standard deviations of 0.02 and 0.04 for bovine and porcine gelatins, respectively, indicating the high accuracy of this method. Trypsin-catalyzed 18O labeling was proved to be an excellent internal calibrant for gelatins. When combined with HPLC and high-resolution mass spectrometry, it is an accurate and sensitive quantitative method for gelatin in the food industry.