Article pubs.acs.org/JAFC
Isolation and Identification of Dipeptidyl Peptidase IV-Inhibitory Peptides from Trypsin/Chymotrypsin-Treated Goat Milk Casein Hydrolysates by 2D-TLC and LC−MS/MS Ying Zhang,† Ran Chen,† Huiqin Ma,‡ and Shangwu Chen*,† †
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 S Supporting Information *
ABSTRACT: 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 firstand 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 DPPIV-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. KEYWORDS: dipeptidyl peptidase IV inhibitor, goat milk, casein, thin-layer chromatography, bioactive peptide
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INTRODUCTION Diabetes is a chronic metabolic disorder that is one of the most prevalent and fastest growing diseases worldwide; the number of people affected by this disease is anticipated to reach 366 million by 2030.1 A new therapy for the management of type 2 diabetes (accounting for 90%−95% of all diabetes cases) focusing on modulation of the incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) has attracted much attention over the past decade.2,3 GIP and GLP-1 are peptidic hormones with glucose-dependent insulin secretion promotion and β-cell-proliferative effect, but with a rather short half-life due to their degradation by dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5, a peptidase responsible for the cleavage of Xaa-Pro- peptide, where Xaa represents an amino aside).2,3 DPP-IV inhibitors can be used to reduce DPP-IV activity and increase the lifetime of incretins, and many natural dietary protein-derived DPP-IV-inhibitory peptides, from egg,4 fish,5 milk,6 rice,7 and amaranth8 proteins, have been reported. Cow milk casein was found, by in silico analysis, to be the richest potential source of DPP-IV-inhibitory peptides among 34 food proteins.9 Several DPP-IV-inhibitory peptides have been isolated and characterized from bovine casein. LPQNIPPL (β-casein, f70−77)10 and FLQP (β-casein, f87−90)11 inhibit DPP-IV with half maximal inhibitory concentrations (IC50) of 46 and 65.3 μM, respectively; VPLGTQ (αs1-casein, f168−174) and EMPFPK (β-casein, f108−114) have relative potencies of 55% and 12%, respectively, of the reference inhibitor diprotin A.12 Compared with cow milk, goat milk possesses unique © XXXX American Chemical Society
functions and is considered to be beneficial for the treatment of diabetes, as stated in the ancient Chinese Compendium of Materia Medica, albeit without detailed explanation.13 Our previous investigations have shown that goat milk casein hydrolysates possess weakly but significantly higher potency than their bovine counterparts in release of DPP-IV-inhibitory peptides, and a commercial trypsin (containing certain chymotrypsin) generated hydrolysate displays the strongest efficiency among five proteases (unpublished results). However, novel and disparate DPP-IV-inhibitory peptides from the goat casein hydrolysates have never been isolated and identified by virtue of a series of separation and identification techniques. Column chromatographies, such as gel chromatography, ionexchange chromatography, and reverse-phase high-performance liquid chromatography (RP−HPLC), are the most common and effective techniques for the separation of bioactive peptides on the basis of size, charge, or polarity and have been widely used.5,7,14 Thin layer chromatography (TLC), as a convenient, simple, fast, and cost-efficient separation technique, is also an indispensable tool for the separation of complex mixtures in analytical chemistry,15 although it has begun to lag behind column chromatography in popularity due to the latter’s more easily automatic operation and convenient interface ability with identification techniques such as mass spectrometry. TLC used Received: June 20, 2015 Revised: August 30, 2015 Accepted: September 1, 2015
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DOI: 10.1021/acs.jafc.5b03062 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
vertical chamber (XinYi, Shanghai, China). The control sample for this first-dimension TLC was spotted on the horizontally opposite side of the plate (1.5 μL SF-0). After the first-dimension separation, the developed plate was dried in air at room temperature to remove eluent and turned 90°; the second-dimension TLC development was carried out at a 90° angle to the first-dimension TLC development with another type of solvent (n-butanol/acetic acid/water, 4:1:1, v/v/v) until the solvent front reached ca. 7.5 cm. The control sample (1.5 μL of SF-0) for the second-dimension TLC was similarly located at the horizontally opposite side. After air-drying at room temperature, 0.05% (w/v) ninhydrin in ethanol was sprayed onto the plate, and peptide coloration was developed at 95 °C for 15 min. Semipreparation of Peptide Fractions and/or Subfractions by TLC. The semipreparative 2D-TLC process was conducted by two coupled silica gel 60 plates with the same solvents and development order as above. The SF-0 sample was first fractionated by the firstdimension TLC, and the recovered fraction was transferred to a new TLC plate for the second-dimension separation. Briefly, 2.5 μL of SF-0 (100 mg/mL) was spotted onto a TLC plate with 12 repeats along the starting line (2 spots were used for peptide visualization, and the other 10 spots were used for semipreparation of the fractions after TLC development with the first- or second-dimension development solvents). Thus, 2.5 mg of SF-0 was loaded and separated on the first-dimension TLC plate. Separated peptide bands were scraped according to the Rf value of the ninhydrin-colored spots, and the silica powders were extracted twice with ddH2O at 20 °C for 20 min (oscillation with an interval of 4 min) to recover the adsorbed peptides completely, then centrifuged at 12 000 × g at 4 °C for 20 min (repeated twice) to exclude the silica. The supernatant for each fraction or subfraction was adjusted to 500 μL as the initial volume of SF-0 (equivalent to 5 mg/mL concentration), and then the DPP-IVinhibitory activity was assayed. RP−HPLC Verification. The peptide samples from the fractions or subfractions of the first- or second-dimension semipreparative TLC were analyzed by RP−HPLC, performed on a Chuangxin Tongheng LC3050 HPLC system. A 20-μL aliquot of sample dissolved in ddH2O was injected into an analytical C18 column with 250 × 4.6 mm i.d., 5 μm, 100 Å (Chuangxin Tongheng Science and Technology Co. Ltd., Beijing, China), and eluted with 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B) as follows: 10% B, followed by a linear gradient from 10 to 50% B from 5 to 45 min, at a flow rate of 1 mL/min at 30 °C. The elution was monitored at 215 nm with a UV/ vis detector; the peptide peaks were collected when needed, and the separation was repeated multiple times to obtain enough samples for analysis. After removing acetonitrile by evaporation, each subfraction was also adjusted to the same initial volume as SF-0 (500 μL at 5 mg/ mL concentration) for assaying their effect on DPP-IV activity as the semipreparative 2D-TLC. Identification of Peptide Sequences by Liquid Chromatography−Tandem Mass Spectrometry. The peptide subfractions with the most efficient DPP-IV-inhibitory activities were subjected to sequence identification by LC−ESI−MS/MS (ESI = electrospray ionization) using a nanoAcquity nano HPLC system (Waters, Milford, MA) coupled to Q-Exactive high-resolution mass spectrometer (Thermo Scientific, Waltham, MA). The sample was injected into a trap column of 2 cm × 100 μm i.d. fused silica capillary (Polymicro, Phoenix, AZ) and separated on a 10 cm × 50 μm i.d. microanalytical column of the same material, which was packed with C18 stationary phase of Aqua 5 μm, 125 Å, and Aqua 3 μm, 125 Å, respectively (Phenomenex, Torrance, CA). The eluting program was conducted with 0.1% FA in water (solvent A) and 0.1% FA in acetonitrile (solvent B) following a linear gradient from 1 to 40% B from 0 to 40 min, at a flow rate of 200 nL/min. The eluate was directly injected into the MS system. A spray voltage of 2000 V was applied to the electrospray tip; the capillary temperature was 320 °C. MS analysis was operated with the following scan parameters: data-dependent MS2 and positive ion mode, full scan mass range 100−1500 m/z, full scan resolution 70 000, MS/MS scan resolution 17 500, mass selection window 4 Da, dynamic exclusion 10 s. The 10 most intensive peptide signals from the full scan were selected for MS/MS scans. The MS/MS data were preprocessed
in the investigation of peptides could provide information about the presence of an analyte or evaluate the general constitutions of a peptide mixture based upon retardation factor (Rf value);16−18 it also was used for the separation and/or identification of certain peptides with known characteristics in combination with other analytical techniques.15,16 However, to the best of our knowledge, there have been no reports on the fractionation and isolation of the more effective components from complex protein hydrolysates, mainly by means of the common silica TLC, for the identification of potent bioactive peptides. Therefore, the objective of this work was to develop a twodimensional silica TLC (2D-TLC) technique using two coupled plates with different eluents to separate and divide the trypsin/chymotrypsin-treated goat casein hydrolysates into fractions with different potencies of DPP-IV inhibition, and to verify its feasibility and efficiency by RP−HPLC at different stages. The more efficient DPP-IV-inhibitory components were then isolated and their peptidic inhibitors were identified.
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MATERIALS AND METHODS
Materials. Skim milk powder from Saanen goat (Capra hircus) was provided by Ausnutria Hyproca Dairy Group BV (Changsha, China). Trypsin (from porcine pancreas, with trypsin ≥250NF U/mg, chymotrypsin ≤75NF U/mg) was from Amresco LLC (Solon, OH). DPP-IV (from porcine kidney, ≥10 U/mg protein) and Gly-Pro-pnitroanilide (H-Gly-Pro-pNA HCl) were purchased from SigmaAldrich (St. Louis, MO). Ile-Pro-Ile (diprotin A) was prepared by Qiangyao Biological Technology Co. Ltd. (Shanghai, China). Chloroform, 25% ammonia, n-butanol, and acetic acid were from Beijing Chemical Works (Beijing, China). Acetonitrile, methanol, trifluoroacetic acid (TFA), and formic acid (FA) were HPLC-grade and from Fisher Scientific (Fair Lawn, NJ). All other chemicals were of analytical grade. Preparation of Hydrolysates. Goat milk casein was prepared by the acidic precipitation method, in which pH is adjusted to the isoelectric point of goat casein, 4.2. The obtained casein precipitate was lyophilized by LGJ-12 vacuum freeze-dryer (Songyuan Huaxing Technology Development Co. Ltd., Beijing, China), and the freezedried casein powder consisted of 95.2 ± 1.1% protein. The casein powder was dissolved in 0.05 M NaOH to 5% (w/w, protein basis) and stirred until complete dissolution; then the solution was preincubated at enzymatic temperature for 10 min, and the hydrolysis was initiated by adding commercial trypsin with a 3% (w/w) substrateto-enzyme ratio and conducted at pH 8.0 and 37 °C for 3 h, with constant temperature and pH (holding with 1 M NaOH). After hydrolysis, the resulting hydrolysates were heated in boiling water for 10 min to inactive the enzyme, then cooled with ice water bath and centrifuged with TGL-20 M high-speed centrifuge (Pingfan Instrument Co. Ltd., Changsha, China) at 1800 × g (4 °C) for 20 min. The supernatant was adjusted to pH 8.0, sequentially filtered through a 0.22-μm syringe filter (Jinteng Corporation, Tianjin, China) and Amicon Ultra-15 centrifugal filter device (Millipore Corporation, Bedford, MA) with molecular-mass cutoff of 5 kDa. The
