Isolation and Identification of Dipeptidyl Peptidase IV-Inhibitory Peptides from Trypsin/Chymotrypsin-Treated Goat Milk Casein Hydrolysates by 2D-TLC and LC-MS/MS.
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.
Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University , Beijing 100083, P. R. China.
Ying
Y
Zhang
Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University , Beijing 100083, P. R. China.
Ran
R
Chen
College of Agriculture and Biotechnology, China Agricultural University , Beijing 100193, P. R. China.
Huiqin
H
Ma
Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University , Beijing 100083, P. R. China.
Shangwu
S
Chen
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry*; D002853:Chromatography, Liquid; D002855:Chromatography, Thin Layer / Q000379:methods*; D002918:Chymotrypsin / Q000737:chemistry; D018819:Dipeptidyl Peptidase 4 / Q000032:analysis; D054873:Dipeptidyl-Peptidase IV Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D006041:Goats; D006868:Hydrolysis; D008892:Milk / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
D002364:Caseins; D054873:Dipeptidyl-Peptidase IV Inhibitors; D010455:Peptides; C475729:casein hydrolysate; D018819:Dipeptidyl Peptidase 4; D002918:Chymotrypsin; D014357:Trypsin
bioactive peptide; casein; dipeptidyl peptidase IV inhibitor; goat milk; thin-layer chromatography