Zhang Ying, Chen Ran, Ma Huiqin, Chen Shangwu
Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University , Beijing 100083, P. R. China.
College of Agriculture and Biotechnology, China Agricultural University , Beijing 100193, P. R. China.
J Agric Food Chem. 2015 Oct 14;63(40):8819-28. doi: 10.1021/acs.jafc.5b03062. Epub 2015 Sep 16.
New dipeptidyl peptidase IV (DPP-IV)-inhibitory peptides from trypsin/chymotrypsin-treated goat milk casein hydrolysates were isolated and identified by two-dimensional silica thin-layer chromatography (2D-TLC) combined to nano LC-MS/MS. 2D-TLC with chloroform/methanol/25% ammonia (2:2:1) and n-butanol/acetic acid/water (4:1:1) as the first- and second-dimension eluents, respectively, in analytical and semipreparative scales, was set up and verified by reversed-phase high-performance liquid chromatography (RP-HPLC) to be feasible and efficient to separate the hydrolysates. Five new DPP-IV-inhibitory peptides, four relatively large oligopeptides (MHQPPQPL, SPTVMFPPQSVL, VMFPPQSVL, and INNQFLPYPY), and AWPQYL were identified, and INNQFLPYPY showed a notable IC50 value of 40.08 μM as an uncompetitive inhibitor. Interactive effects on DPP-IV inhibition were also observed among separated fractions and pure synthetic peptide mixtures with concentration-dependent activity. The study gives new insights into goat casein hydrolysates with identified DPP-IV-inhibitory peptides efficiently isolated by 2D-TLC, which provides a simple and cost-efficient separation process and is compatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification.
通过二维硅胶薄层层析（2D-TLC）结合纳米液相色谱-串联质谱（nano LC-MS/MS），从胰蛋白酶/胰凝乳蛋白酶处理的山羊奶酪蛋白水解物中分离并鉴定出新型二肽基肽酶IV（DPP-IV）抑制肽。分别以氯仿/甲醇/25%氨水（2:2:1）和正丁醇/乙酸/水（4:1:1）作为第一维和第二维洗脱剂，建立了分析级和半制备级的2D-TLC方法，并通过反相高效液相色谱（RP-HPLC）验证其对于分离水解物是可行且高效的。鉴定出了5种新型DPP-IV抑制肽、4种相对较大的寡肽（MHQPPQPL、SPTVMFPPQSVL、VMFPPQSVL和INNQFLPYPY）以及AWPQYL，其中INNQFLPYPY作为非竞争性抑制剂表现出显著的IC50值，为40.08 μM。在分离的组分和具有浓度依赖性活性的纯合成肽混合物之间也观察到了对DPP-IV抑制的相互作用。该研究为通过2D-TLC有效分离出具有已鉴定的DPP-IV抑制肽的山羊酪蛋白水解物提供了新见解，2D-TLC提供了一种简单且经济高效的分离方法，并且与液相色谱-串联质谱（LC-MS/MS）鉴定兼容。
