Nippi Research Institute of Biomatrix , 520-11 Kuwabara , Toride , Ibaraki 302-0017 , Japan.
Department of Nutrition, Faculty of Domestic Science , Tokyo Kasei University , 1-18-1 Kaga , Itabashi-ku, Tokyo 173-8602 , Japan.
J Agric Food Chem. 2019 Apr 24;67(16):4671-4678. doi: 10.1021/acs.jafc.9b00571. Epub 2019 Apr 9.
Collagen-derived hydroxyproline (Hyp)-containing oligopeptides, known to have various physiological functions, are detected in blood at markedly higher concentrations after oral ingestion of collagen hydrolysate. Monitoring the absorption and metabolism of the bioactive peptides is essential to investigate the beneficial effects of collagen hydrolysate. We previously developed an internal standard mixture by sequential protease digestion of stable isotope-labeled collagen, which enabled highly accurate quantitation of collagen-derived oligopeptides by liquid chromatography-mass spectrometry (LC-MS). However, the use of proteases caused a profound imbalance in the generated peptides. Here, we employed partial acid hydrolysis to achieve more efficient and balanced peptide generation. Various stable isotope-labeled oligopeptides were detected after 0.5 h acid hydrolysis, and marked enhancement of peptide generation compared with the previous enzymatic method was observed, especially for Hyp-Gly (27.8 ± 0.6 ng/μg vs 0.231 ± 0.02 ng/μg). The acid hydrolysate was then heated to generate labeled cyclic dipeptides. Using the novel internal standard mixture in LC-MS, we were able to simultaneously quantitate 23 collagen-derived oligopeptides in human plasma and urine after oral administration of collagen hydrolysate.
胶原蛋白衍生的羟脯氨酸（Hyp）含量的低聚肽，具有多种生理功能，在口服胶原蛋白水解物后，在血液中的浓度明显升高。监测生物活性肽的吸收和代谢对于研究胶原蛋白水解物的有益作用至关重要。我们之前通过稳定同位素标记的胶原蛋白的顺序蛋白酶消化开发了一种内标混合物，通过液相色谱-质谱（LC-MS）实现了胶原蛋白衍生的低聚肽的高度准确定量。然而，蛋白酶的使用导致产生的肽严重失衡。在这里，我们采用部分酸水解来实现更高效和平衡的肽生成。在 0.5 小时酸水解后检测到各种稳定同位素标记的低聚肽，与以前的酶法相比，观察到肽生成的显著增强，尤其是 Hyp-Gly（27.8 ± 0.6 ng/μg 对 0.231 ± 0.02 ng/μg）。然后将酸水解物加热以生成标记的环状二肽。使用新型内标混合物在 LC-MS 中，我们能够在口服胶原蛋白水解物后同时定量人血浆和尿液中的 23 种胶原蛋白衍生的低聚肽。
