Identification and Quantification of DPP-IV Inhibitory Peptides from

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Bioactive Constituents, Metabolites, and Functions

Identification and Quantification of DPP-IV Inhibitory Peptides from Hydrolyzed Rapeseed Protein-Derived Napin, with Analysis of The Interaction between Key Residues and Protein Domains Feiran Xu, Yijun Yao, Xiaoying Xu, Mei Wang, Mengmeng Pan, Shengyang Ji, Jin Wu, Donglei Jiang, Xingrong Ju, and Lifeng Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01069 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 12, 2019

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Journal of Agricultural and Food Chemistry

School of Food Science and Technology, Jiangnan University; College of Food Science and Engineering, Collaborative Innovation Center for Modern Grain Circulation and Safety, Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing University of Finance and Economics Wang, Lifeng; College of Food Science and Engineering, Collaborative Innovation Center for Modern Grain Circulation and Safety, Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing University of Finance and Economics

ACS Paragon Plus Environment


Identification and Quantification of DPP-IV Inhibitory Peptides from Hydrolyzed Rapeseed Protein-Derived Napin, with Analysis of The Interaction between Key Residues and Protein Domains Feiran Xu1; Yijun Yao1, 2; Xiaoying Xu2; Mei Wang2; Mengmeng Pan2; Shengyang Ji2; Jin Wu2; Donglei Jiang2; Xingrong Ju*1,2; and Lifeng Wang*2 1. National Engineering Laboratory for Cereal Fermentation Technology, State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, People's Republic of China. 2. College of Food Science and Engineering, Collaborative Innovation Center for Modern Grain Circulation and Safety, Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing University of Finance and Economics, No. 3 Wenyuan Road, Nanjing, Jiangsu 210023, People’s Republic of China.

*Corresponding Author: Prof. Xingrong Ju, [email protected] Tel: +86 25 8402 8788; Fax: +86 25 8402 8788 Prof. Lifeng Wang, [email protected] Tel: +86-025-86718569; Fax: +86-025-86718569 1


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Abstract

2

Previously reported peptides derived from Napin of rapeseed (Brassica napus) have

3

been shown to inhibit DPP-IV in silico. In the present study, Napin extracted from

4

rapeseed was hydrolyzed by commercial enzymes and filtered by ultrafiltration

5

membrane. Napin hydrolysate purified by Sephadex G-15 gel filtration column and

6

preparative RP-HPLC. The two-enzyme combination approach of Alcalase and

7

Trypsin was the most favorable for the DPP-IV inhibitory activity (IC50=0.68 mg/mL)

8

of the Napin hydrolysate. Three peptides and one modified peptide (pyroglutamate

9

mutation at the N-terminus) were identified using HPLC-Triple TOF MS/MS.

10

DPP-IV inhibitory activity and the enzyme inhibition types of them were also

11

determined. Meanwhile, key residues associated with the interactions between the

12

selected peptides and DPP-IV were investigated by Molecular docking. IPQVS has

13

key amino acid residues (Tyr547, Glu205 and Glu206) that is consistent with Diprotin

14

A. ELHQEEPL could form a better covalent bond with Arg358 in the S3 pocket of

15

DPP-IV.

16 17

Keywords

18

Napin hydrolyzate; Peptides preparation; DPP-IV inhibitory peptides; In vitro;

19

Molecular docking.

20 21 22 2



23

Introduction

24

Type 2 diabetes is one of the major chronic diseases that seriously affects humans

25

health worldwide, and it accounts for more than 90% of patients with diabetes and

26

result in microvascular and macrovascular complications.1, 2 The number of people

27

suffering from Type 2 diabetes is expected to reach 439 million by 2030.2

28

Unavoidable, many hypoglycemic drugs can cause side effects, such as acarbose,

29

sulfonylureas. Thus, developing new natural hypoglycemic drugs with low side

30

effects is essential for food and medical researchers.

31

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide

32

(GIP) can promote insulin secretions, suppress pancreatic glucagon release, and

33

reinforce β-cell proliferative effects.1 However, GLP-1 and GIP are rapidly degraded

34

by DPP-IV in the organ or intestinal epithelium, and their half-life period is only 1 to

35

2 minutes.3 The inactivation and reduction of DPP-IV can maintain sufficient content

36

levels of GLP-1 and GIP in the body. The goal of DPP-IV inhibitors is to prolong

37

GLP-1 and GIP action, which regulates blood glucose level by the stimulation of

38

insulin secretion. For this reason DPP-IV inhibitors are among the newest medications

39

that have been introduced to the type 2 diabetes therapies.4 The synthetic DPP-IV

40

inhibitors sitagliptin and Diprotin A currently cause a 2-fold increase in endogenous

41

GLP-1 levels for the treatment of type 2 diabetes.5,

42

pharmaceutical inhibitors of DPP-IV can be used as a first-line alternative treatment

43

to reduce hyperglycemia and hemoglobin A1C levels in the case of metformin

44

intolerance or contraindications.7-9

6

It is more advantageous that

3


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Nowadays, plenty of works have focused on the strategy to identify natural peptide

46

inhibitors of DPP-IV activity as a substitute for synthetic drugs. Peptides derived from

47

food proteins have been proved to inhibit DPP-IV in silico, in vitro and in vivo. For

48

example, the α-lactalbumin hydrolysate obtained by peptic digestion of whey protein

49

has the greatest potency with an IC50 value of 0.036 mg/mL.10 Hou et al. found that

50

RRDY from yam dioscorin hydrolysis lowers the area under the curve (AUC0−120) of

51

blood glucose and DPP-IV activity in normal ICR mice.11 Octapeptide (LQAFEPLR)

52

and nonapeptide (EFLLAGNNK) derived from oat 11S globulin have been Identified,

53

and their DPP-IV inhibiting activities have been determined.12 Silver carp protein

54

(SCP) hydrolysates contain efficacious DPP-IV inhibitory peptides which have been

55

investigated via in silico hydrolysis analysis, peptide separation combined with liquid

56

chromatography−tandem mass spectrometry (LC−MS/MS) identification, and

57

chemical synthesis.13 Interestingly, an in silico approach has been reported to predict

58

the potential DPP-IV inhibitory peptides derived from 72 dietary proteins.14 Among

59

these 72 dietary proteins, three kinds of plant proteins were endued with the best

60

inhibitory properties, including Palmaria palmata (allophycocyanin a chain and

61

phycoerythrin b subunit), rapeseed (Napin small chain), and wheat (glutenin, low

62

molecular weight subunit 1D1).14 Furthermore, the DPP-IV inhibitory potency index

63

(PI) of these proteins is greater than 5.00×10-6 μM-1g-1, and the protein coverage (PC)

64

of these proteins is 16.9%, 16.7%, and 15.8%, respectively.14 Napin separated from

65

rapeseed (Brassica napus) has been identified as a potential and rich source of

66

DPP-IV inhibiting peptides. More particularly, hydrolysates and peptides derived 4



67

from Napin protein have not been experimentally evaluated for their in vitro DPP-IV

68

inhibition properties.

69

Napin (Accession number: P17333) is one of the major rapeseed storage proteins

70

(the other one is Cruciferin).15 With an isoelectric point (PI) of approximately 11,

71

Napin is particularly basic.16 In terms of elementary structure, Napin is composed of

72

two polypeptide chains with molecular weights of 9-10 kDa and 4-4.5 kDa that are

73

held together mainly by two disulfide bonds. Additionally, two additional intrachain

74

disulfide bonds in the large chain reinforce the stability of the proteins.17 Because 2S

75

albumin (rich in Napin) has emulsifying properties, gelling properties and low content

76

of anti-nutrients, it is was widely used in the food industry products, such as

77

mayonnaise and desserts.16 Hence, discoveries of hypoglycemic peptides in Napin

78

will continue to drive its development for healthy foods derived from abandoned

79

proteins. In addition, our laboratory has performed many studies on the identification

80

of rapeseed active peptides18-20, and the DPP-IV inhibitory activity of Napin peptides

81

will be another important breakthrough.

82

The objectives of the present study were to prepare and evaluate DPP-IV inhibitory

83

peptides derived from Napin. First, bioassay-guided, biochemical fractionation, and

84

HPLC-Triple-TOF MS/MS were used to identify the peptides within selected

85

hydrolysates. Second, Molecular docking approaches were used to better evaluate the

86

mechanisms of DPP-IV inhibition by Napin-derived peptides. Besides these, the type

87

of enzyme (DPP-IV) inhibition of different appraised peptides was determined by the

88

enzyme kinetics assay. 5


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Materials and Methods

90

Chemicals and Materials

91

DPP-IV (EC 3.4.14.5, come from human) and Gly-Pro-p-nitroanilide were

92

purchased from Sigma-Aldrich (St. Louis, MO). Diprotin A (Ile-Pro-Ile, ≥95% purity)

93

was purchased from Qiangyao Biological Technology Co., Ltd. (Shanghai, China).

94

HPLC grade acetonitrile and trifluoroacetic acid (TFA) were purchased from Merck

95

Co., Ltd. (Darmstadt, Germany). Pepsin, Trypsin, Flavourzyme, Papain, Tris-HCl,

96

β-mercaptoethanol, sodium dodecyl sulfate (SDS), and broad range MW marker

97

(5-245kDa) were procured from Solarbio Biological Technology Co., Ltd. (Beijing,

98

China). Purified water was used a Milli-Q System (Millipore Corporation, Milford,

99

MA, USA). All other chemicals were of analytical grade. Rapeseeds (Brassica napus)

100

were obtained from the Institute of Food Science and Technology CAAS (Beijing,

101

China). Identified peptides (> 95% purity, MPGPS, PAGPF, TMPGP, IPQVS,

102

NIPQVS,

103

Q(-17.03)KTMPGP) were synthesised by solid-phase method using the FMOC

104

synthesis from Bank Peptide Ltd. (Hefei, China).

105

Preparation of Napin Protein

KTMPGPS,

HQEEPL,

ELHQEEPL,

Q(-17.03)QWLH,

and

106

Napin was prepared using the method described by Wu et al.17 and Apenten et al.21

107

with modifications. First, rapeseeds were shelled and ground into powder using a

108

laboratory mill with a screen size of 0.5 mm. Prior to use as protein isolates, rapeseed

109

powder samples were deoiled using petroleum ether. In the second experiment,

110

powder samples were mixed with Tris-HCl buffer (0.05 M, pH 7.0) containing 0.1 M 6



111

NaCl (200 mL) for 2 h at room temperature. The slurry was centrifuged at 15,000×g

112

and 4 °C for 20 min. The supernatant was adjusted gradually (in 15 min) to pH=4

113

with a 0.1 M HCl solution, and the resultant slurry was centrifuged using the same

114

conditions as above. Finally, ultrafiltered protein isolate was recovered from the acid

115

supernatant by an ultrafiltration system (Millipore, Bedford, MA, US) with a

116

molecular weight cut-off at 10-30 kDa. After being dialyzed against deionized water

117

overnight, Napin was lyophilized and stored at -20 °C for further use.

118

Characterization and Quantification of Napin

119

According to the manufacturer’s instructions and refer to the method of Li-Chan et

120

al.10 with modifications, SDS-PAGE was performed using a Bio-Rad apparatus, using

121

ready-made gels and ready-made buffer strips (16.5% Tricine gel). A mixture of

122

proteins standards (5-245 kDa) was used as a broad range MW marker. Each sample

123

was dissolved in 0.0625 M Tris–HCl buffer (pH 7.4) containing 2% (w/v) SDS, 4 M

124

urea and 0.1 M 2-mercaptoethanol. The samples were heated at 100 °C for 10 min and

125

subjected to electrophoresis and stained with Coomassie Brilliant Blue G-250.

126

The molecular weight distribution and purity of Napin were estimated by high

127

performance size-exclusion chromatography (SEC-HPLC) using an Agilent 1100

128

instrumentation equipped with a TSK gel 2000 SWXL column (300×7.8 mm, Tosoh,

129

Tokyo, Japan). Napin treated with β-mercaptoethanol was applied to the column,

130

eluted at a flow rate of 0.5 mL/min and monitored at 220 nm and 30 °C by isocratic

131

elution. The composition of solvent was acetonitrile/ ultrapure water/ rifluoroacetic

132

acid (45:55:0.1). A molecular weight calibration curve was prepared from the average 7


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retention times of the following standards: bovine serum albumin (MW 67000 Da),

134

cytochrome C (MW 12500 Da), rapeseed peptide (MW 1158.57 Da), glutathione

135

(MW 307.32 Da) and glycine (MW 75 Da) (Sigma Co., St. Louis, MO, USA).

136

Preparation of Napin Hydrolysates

137

To produce DPP-IV inhibitory peptides for further study, hydrolysis was conducted

138

using a step-by-step enzymatic method that included four selections of enzyme

139

described in Table 1. The method of preparation of Napin hydrolysates was

140

performed according to a previous method.12 The pH of each enzymatic procedure

141

was adjusted to the working value of selected enzyme using 1.0 M NaOH and HCl

142

and reacted with the enzyme at respective temperature for 5.0 h. In addition, the

143

hydrolysis performed using the enzyme/substrate ratio listed in Table 1. After

144

hydrolysis, the acquired hydrolysates were heated in water (95 °C) for 10 min to

145

inactivate the enzyme, then cooled with ice and centrifuged at 13,000g for 30 min. A

146

portion of the supernatant containing target peptides was passed through ultrafiltration

147

membranes with molecular weight cut-off (MWCO) of 1 and 3 kDa by an

148

ultrafiltration system (Millipore, Bedford, MA, US). The permeate from each MW

149

membrane was collected as 3 kDa peptide fractions, respectively. The

150

protein concentration in the supernatant was determined by the Folin-phenol method

151

using BSA as a standard, and then freeze-dried and stored at -20 °C prior to use.

152

Protein content in raw materials was determined using the Kjeldahl method, which is

153

referred to as the national standard. The protein content in the Napin hydrolysate was

154

determined using the Folin-phenol method. 8



155

Degree of Hydrolysis (DH%) Determination

156

The DH % which is defined as the percentage of peptide bonds cleaved, was

157

calculated from the volume and molarity of NaOH used to maintain the pH constant

158

(Equation 1).22 The Napin hydrolysate was diluted 400 times with deionized water for

159

the determination of hydrolysis percentage. (1)

160 161

where B is the amount of NaOH consumed (mL) to maintain the pH value constant

162

during the proteolysis of the substrate. Nb is the normality of the base, MP is the mass

163

(g) of the protein (N × 6.25), and α represents the average degree of dissociation of

164

the α-NH2 groups in the protein substrate, h is the total number of peptide bonds in the

165

protein substrate (7.8 mmol/g for rapeseed protein).

166

Purification of DPP-IV Inhibitory Peptides

167

Napin hydrolysate (Alcalase+Typsin) was suspended in 5 mL of deionized H2O and

168

then loaded onto a Sephadex G-15 gel filtration column (3.0cm×200 cm) equilibrated

169

with 0.2M sodium acetate buffer (pH=4). The column was then eluted with the same

170

solution, and fractions were collected at a flow rate of 30 mL/h. The resultant fraction

171

exhibiting the highest DPP-IV inhibitory activity was further purified using a

172

preparative RP-HPLC on a Shodex RI-201H instrumentation. Each eluted peak from

173

the preparative liquid phase was collected, concentrated by vacuum rotary

174

evaporation, and lyophilized to obtain each separated component. The injection

175

volume was changed to 200 μL, and the flow rate was adjusted to 10 mL/min. With

176

the remaining elution conditions unchanged, the separated peptide fractions were 9


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collected using a semi-preparative HPLC (Waters Technologies, Milford, MA, USA)

178

equipped with a diode array detector (DAD) and a quarternary pump. A Waters

179

preparative C18 column (PrepHT, Zorbax300SB-C18, 2×250mm) maintained at 30

180

°C was used.

181

Identification of Peptide Sequences by HPLC-Triple TOF MS/MS

182

The resultant fraction exhibiting the highest DPP-IV inhibitory activity after

183

purification was analyzed by HPLC-Triple TOF MS/MS using a method described in

184

Xu19,

185

equipped with a 4.6 × 150 mm C18 5μm reversed-phase column (DIONEX USA). A

186

25 μL aliquot of the sample was injected using an autosampler.

187

TFA (v/v) in water, and solvent B was 0.1% TFA (v/v) in acetonitrile. The flow rate

188

was 1.0 mL/min, and a variable wavelength absorbance detector was set at 220 nm.

189

The gradient sequence was as follows: 0% B from 0 to 5 min, 60−10% B from 5 to 25

190

min, and 10−0% B from 25 to 30 min. Advanced mass spectrometry was performed

191

on a Triple TOF 5600+system (AB SCIEX, CA, USA) with a Duo-Spray source

192

operating in positive ESI mode. Optimized parameters were used, with slight

193

modification.20

20

with some modifications. Agilent 1100 instrumentation was used and

Solvent A was 0.1%

194

According to Caron et al.23, MS/MS data were processed by PEAKS Studio Version

195

7 (Bioinformatics Solutions Inc., Waterloo, Canada) using UniProtKB entries

196

(Accession number: P17333). The peptide identity search was performed by selecting

197

the Alcalase + Trypsin enzymes with a maximum of 100 missed cleavages allowed.

198

Assessment of the DPP-IV Inhibitory Activity by Hydrolysates and 10



199

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Peptides

200

The DPP-IV inhibition assay was carried out as described by FitzGerald et al.24 and

201

Cudennec et al.25 with a slight modification. The assay was performed in 96-well

202

plates using Diprotin A as a positive control for 1 h at 37 °C. Test samples (50μL)

203

were contained the reaction substrate Gly-Pro-p-nitroanilide (final concentration of

204

0.2 mM). The negative control contained 100 mM Tris–HCl buffer pH 8.0 (50μL) and

205

the reaction substrate. The reaction was initiated by the addition of DPP-IV (final

206

concentration of 0.0025 units/mL). All the reagents and samples were diluted in 100

207

mM Tris–HCl buffer (pH=8.0). Plates were read at 405 nm using a SpectraMax M2e

208

Microplate Reader (Molecular Devices Inc., San Francisco, CA, USA). The DPP-IV

209

IC50 values (concentration of active compound required to observe 50% DPP-IV

210

inhibition) were determined by plotting the percentage of inhibition as a function of

211

the concentration of test compound. The data of Napin hydrolysates purified by

212

preparative liquid phase were expressed as percent of remaining activity in the

213

presence of test samples versus control.

214

(2)

215

where A405 (sample) is the absorbancy at 405 nm (OD405) in the presence of DPP-IV,

216

reaction substrate (Gly-Pro-p-nitroanilide), and peptide sample; A405 (sample/control)

217

is the OD405 when DPP-IV is substituted with Tris-HCl buffer (100 mM, pH 8.0) as

218

sample assay; A405 (negative/reaction) is the OD405 of DPP-IV and reaction substrate

219

with no inhibitor; and A405 (negative/control) is the OD405 of the assay with no

220

DPP-IV or inhibitor. 11


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221

The IC50 values were calculated with the linear regression equation obtained after the

222

appropriate concentrations of hydrolysates and peptides assayed against the DPP-IV

223

% of inhibition.

224

Mode of Inhibition of DPP-IV

225

According to FitzGerald et al.24, the mode of inhibition of the different compounds

226

was investigated using Lineweaver and Burk analysis by measuring the initial rate of

227

the reaction at different Gly-Pro-p-nitroanilide concentrations ranging between 0 and

228

100 μM without inhibitors and in the presence of peptides at their IC50 concentrations.

229

Km and Vmax values were deducted from the Lineweaver and Burk double reciprocal

230

plots. (3)

231

(4)

232 233

Molecular Docking Analysis

234

In order to obtain the spatial conformation, DPP-IV inhibitory peptides with IC50

235

values less than 200μM were refined using MD simulation. Docking of the four

236

peptides optimized by MD to the binding site of DPP IV (PDB ID: 4PNZ) was

237

performed using MOE. The docking pocket was consistent with published articles.26

238

Accelrys Discovery Studio 4.0 (Accelrys Software Inc.,) was used to strike out water

239

molecules, and optimize the spatial structure of the peptides. The produced

240

conformation with minimum binding energy was selected for the analysis. FF12SB

241

force fields were employed for the peptides. Prior to the production process, the

242

whole system was subjected to minimization and equilibrium in NVT and NPT, 12



243

respectively. Afterwards, a 20 ns non-restrained molecular dynamics simulation was

244

then performed in NPT as the final production phase. Details for setting up of the MD

245

simulations have been described by Sansom et al.27. MD trajectories were analyzed

246

using VMD, and the molecular structures were generated using PyMol (Schrodinger,

247

LLC). The score of the final and stable peptide-substrate complex was assessed using

248

the total interaction energy between the active site and the substrate.

249

Statistical Analysis

250

All experiments were performed at least three times. Statistical analysis was

251

performed using GraphPad Prism V using one-way and two-way ANOVA to

252

determine significant differences between means (p85%) were 10.25

270

kDa and 5.11 kDa which were consistent with other literature reports.15, 16 There was

271

a significant difference between line 1 and line 2, indicating that the Napin protein

272

was enriched by the improved ultrafiltration system used in the isolation process.

273

Molecular weight distributions of protein were accurately identified by the TSK gel

274

column, importantly, the high-purity Napin provided a preliminary basis for the

275

separation of subsequent DPP-IV inhibiting peptides.

276

Hydrolysis of Napin Protein and DPP-IV Inhibitory Activities of

277

Hydrolysates

278

Under the condition of Alcalase + Typsin, the hydrolysate derived from Napin had

279

the strongest DPP-IV inhibiting activity (IC50=0.68mg/mL) (Table 1). The present

280

(see Table 1) showed that the degree of hydrolysis of Napin hydrolysate under

281

Trypsin treatment was not optimal (DH=15.06±2.70%), but did not affect its highest

282

DPP-IV inhibitory activity. Indeed, hydrolysates with MWs less than 1 kDa displayed

283

greater inhibition capacity of DPP-IV than other ultrafiltration components with IC50

284

values ranging from 0.68 to 1.87 mg/mL (Table 1). The result in this study is in

285

agreement with former studies that found the preferable DPP-IV-inhibitory peptides

286

derived from food protein consisted of two to ten amino acid residues. Several studies 14



287

have demonstrated that the DPP-IV inhibitory activity of hydrolysate is closely

288

related to the type and cooperation of protease. For example, Zhang et al. obtained

289

optimal DPP-IV inhibitory peptides from Trypsin/Chymotrypsin-treated goat milk

290

casein hydrolysates;28 and the α-lactalbumin hydrolysate prepared by Pepsin treatment

291

has the greatest potency with an IC50 value of 0.036 mg/mL.10 Trypsin is more

292

beneficial for the release of DPP-IV inhibitory peptide in camel milk protein in silico,

293

and the peptides LPVP and MPVQA are considered stable.29 Instead, sequential

294

enzymatic hydrolysis with Flavourzyme following Alcalase treatment improved the

295

DH value. We infer that the first enzymatic step using Alcalase allowed many peptide

296

bonds in Napin to be accessible because it specifically recognizes the Trp-, Tyr-, Leu-,

297

Ala-, and Val- sites and does not affect the Pro- amino acid residues in the

298

hydrolysate. Of note, the Trypsin cleavage site is precisely for basic amino acids that

299

account for the majority of amino acid residues in DPP-IV inhibitory peptides.30

300

Extensive hydrolysis such as Flavourzyme may further hydrolyze the obtained

301

bioactive sequence of peptides and thereby decrease their DPP-IV inhibitory activity.

302

Lower molecular weight of hydrolysates generally have better biological activity in

303

respect of antioxidation,19 ACE inhibition,31, 32 and tumor cell inhibition activities,18

304

including inhibition of DPP-IV activity. These results confirmed that the DPP-IV

305

inhibitory activity of Napin-derived peptides is strongly influenced by the protease

306

used in the process of enzymatic hydrolysis and molecular weight of hydrolysate.

307

Isolation and Purification of DPP-IV Inhibitory Peptides Derived

308

from Napin Protein 15


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309

From the results of the previous chapter, hydrolysates prepared from Alcalase +

310

Typsin was selected for further identification of sequence of amino acid residues to

311

screen novel DPP-IV inhibiting peptides. The DPP-IV inhibiting peptides in Napin

312

hydrolyzates were isolated by a molecular exclusion chromatography method. The < 1

313

kDa MW fraction was loaded on a size exclusion gel chromatography column (10 mm

314

× 1000 mm) using diluents of Sephadex G-15 gel. As shown in Fig 2A, three fractions

315

(G1, G2, and G3) were obtained and collected separately. Among them, the

316

DPP-IV-inhibition activity of fraction G2 had the lowest IC50 value (0.25mg/mL),

317

which was close to one-fifth of that of fraction G1 (Fig 2B). The mass spectrometry

318

TIC image of the hydrolysate after purification by Sephadex G-15 gel is shown in Fig

319

2C. It can be found that the distribution of the characteristic peaks was uniform and

320

the composition of polypeptides was complex, suggesting that the DPP-IV active

321

fractions need to be further screened. This potent DPP-IV inhibitory fraction G2 was

322

further isolated and fractionated via preparative RP-HPLC on a Shodex RI-201H

323

instrumentation into four major resultant fractions (Fig 3A). Different from the ion

324

exchange chromatography column and gel column, the purpose of this system was to

325

further separate the target peptides by the difference in intermolecular polarity.33

326

Unfortunately, the two characteristic peaks in fraction R2 were not completely

327

separated because the points of retention time for them were too close. All the

328

fractions in Fig 3B showed DPP-IV inhibitory activity. Fraction R2 exhibited the

329

exceptionally highest inhibition at 1 mg/mL peptide concentration. This test of

330

concomitant inhibitory activity test during separation and purification of peptides has 16



331

been proven to be effective.30,

34

332

successfully obtained from the complex hydrolysate.

To a great extent, the target peptide can be

333

As a follow-up study, Fig 4A shows the HPLC-chromatogram obtained for fraction

334

G2-R2. The main 10 absorption peaks observed, and two of which had significantly

335

higher absorption peaks than the rest. According to the detection of Triple-TOF

336

MS/MS detection and the processing analysis of PEAKS Studio (Version 7), the

337

amino acid residue sequences of 10 peptides were determined and arranged according

338

to their retention time (Fig 4A and Table 2). The cross-repetition of peptide sequence

339

information in rapeseed (Brassica napus) protein data resulted in fractions G2-R2-2,

340

which required de novo sequencing. This phenomenon has also appeared in DPP-IV

341

inhibitory peptides from the macroalga Palmaria palmate.35 Taking into account the

342

existence of the modification of peptides, the remaining 9 peptides were found by

343

PEAKS DB and PEAKS PTM. During the separation and purification process, the

344

N-terminus of fractions M1 and M2 produced modification of the decarbamylation

345

constant (-17.03), which caused irreparable damage to their DPP-IV inhibiting

346

activity. This is mainly due to the fact that the N-terminal structure of the peptide

347

plays a crucial role in its interaction with proteases or proteins.29, 36 Additionally, this

348

phenomenon indicated that commercial enzymes might cause little negative

349

modification while hydrolyzing plant proteins. The molecular interactions between

350

the N-terminal amino acid residue of peptide Q (-17.03) KTMPGP and DPP-IV was

351

discussed in the next Section. Fig 4B shows the mass spectrometry results with the

352

main acquisition peak data of fraction G2-R2 focusing on 3-5 min. The results of 17


Page 18 of 49

Page 19 of 49


353

peptide sequence analysis are shown in Fig 4C: the abbreviated amino acid marked as

354

green represented the enzyme cleavage site (Alcalase + Typsin), and the blue

355

underline represented the sequences of amino acid residue produced after enzyme

356

digestion.

357

Quantification of DPP-IV Inhibitory Activities of Purified Napin

358

Peptides

359

In order to further study the mechanism of DPP-IV inhibitory activity, identified

360

peptides (> 95% purity, MPGPS, PAGPF, TMPGP, IPQVS, NIPQVS, KTMPGPS,

361

HQEEPL, ELHQEEPL, Q(-17.03)QWLH, and Q(-17.03)KTMPGP) were synthesised

362

by solid-phase methodology using the FMOC synthesis. The amino acid residue

363

sequences of 10 peptides derived from Napin are listed in Table 2, and 90% of which

364

contained a Pro residue. These results matched peptides reported in the literature with

365

DPP-IV inhibitory activity.14,

366

inhibitory activity. In particular, three peptides and one modified peptide

367

(pyroglutamate mutation at the N-terminus) showed prominent inhibitory activity

368

with IC50 values of 135.70±8.24μM (PAGPF, 487.2430Da), 162.73 ± 12.26μM (Q

369

(-17.03) KTMPGP, 740.3527Da), 52.16±5.91μM (IPQVS, 542.3064Da), and

370

78.46±4.94μM (ELHQEEPL, 993.4767Da), respectively. The peptide IPQVS

371

contained Pro as the second N-terminal amino acid residue, indicating a greatly

372

probable DPP-IV inhibitory activity.37 Based on the DPP-IV inhibition activity of

373

IPQVS being higher than that of MPGPS (Table 2), the first amino acid residue at the

374

N-terminus may also affect the DPP-IV inhibitory activity of the peptide with the

36

All ten peptides presented in Table 2 had DPP-IV

18



375

same Pro position.38 Significant differences (p < 0.05) were observed for the DPP-IV

376

IC50 values of the peptides HQEEPL and ELHQEEPL(>200μM and 78.46±4.94μM,

377

respectively). It is believed that the first two N-terminal amino acids of peptides

378

dominate their DPP-IV inhibitory properties, while the other amino acids residue play

379

minor role in DPP-IV inhibition.29 Besides, Jin et al. reported that a greater peptide

380

sequence length may also increase the DPP-IV inhibitory activity.39 In contrast with

381

other fractions, the DPP-IV inhibiting activity of the peptide IPQVS proved to be the

382

strongest in vitro. Even with a similar inhibition rate to that of Diprotin A (3.62 ± 0.37

383

μM), the inhibition was not completely confirmed due to the actual reaction in vivo.

384

The RP-HPLC and secondary mass spectrometry analysis of four DPP-IV inhibitory

385

peptides are shown in Fig 5A-D.

386

Mode of Inhibition of Purified Napin DPP-IV Inhibitory Peptides

387

The inducement of inhibition of Napin on key sites of the DPP-IV and the

388

underlying molecular mechanism remain unclear. In this section, we explored the

389

types of inhibitory responses of the four DPP-IV inhibiting peptides. As illustrated in

390

Fig 6, the four peptides were dissolved in ultrapure water at concentrations of 0–100

391

mM, and the type of inhibition were determined from Lineweaver–Burk double

392

reciprocal plots. Corresponding error bars and correlation coefficient are shown in

393

Supporting information for publication (Figure S1). Our results found that the peptide

394

PAGPF and ELHQEEPL showed apparently competitive/ noncompetitive mixed type

395

inhibition of DPP-IV, and the Q (-17.03) KTMPGP peptide showed uncompetitive

396

inhibition of DPP-IV. The peptide IPQVS behaved as a competitive inhibitor. In 19


Page 20 of 49

Page 21 of 49


397

general, the enzymatic products of peptides, such as IPI (Diprotin A) and IPQVS (the

398

N-terminal second position is Pro), continue to exert inhibition because they can be

399

decomposed by DPP-IV. ELHQEEPL is not hydrolyzed by DPP-IV, suggesting that it

400

might be able to enter the internal binding sites of DPP-IV with the original structure.

401

The perfect combination of the peptide with the DPP-IV pocket or active center might

402

have a great inhibitory effect,40 which will be discussed in detail using Molecular

403

docking in the next section. Although the peptide Q (-17.03) KTMPGP did not

404

competitively inhibit DPP-IV, Pro in the C-terminus prompted its value of IC50 also

405

lower than 200μM. Nongonierma et al.41 screened an analogous strongly DPP-IV

406

inhibiting peptide FLQP from casein by an in silico approach. Through these two

407

peptides, we initially confirmed that -Xaa-Pro is also a typical structure with high

408

DPP-IV inhibitory activity. Additionally, this structure can also provide a strong

409

activity of antioxidant (DPPH scavenging) in vitro. 19, 20, 41

410

Analysis of The Interaction Between Key Residues and Protein

411

Domains

412

Molecular docking studies were performed to reveal the key interactions that mimic

413

the production of peptides and to better rationalize their different behaviors. A lower

414

REU of energy score relates to a better molecular binding conformation between

415

peptides and DPP-IV. 42 As shown in Table 3, the molecular modeling parameters of

416

the four peptides are listed in detail. Although Q (-17.03) KTMPGP has many sites

417

for binding to DPP-IV, each of the binding energies was found to be weaker than

418

those of ELHQEEPL, especially the interface energy. Compared to other peptides, 20



419

ELHQEEPL possessed a lower interface energy value (-14.67 REU) and even lower

420

than Diprotin A. ELHQEEPL and DPP-IV had the lowest energy when they

421

interacted and formed a complex regardless of the optimization and correction of

422

initial conformation.43 These data may be explained by the strong interaction between

423

residue Glu (the N-terminus of ELHQEEPL) and Arg358 which in the S3 pocket of

424

DPP-IV (Fig 8B).26, 44 The DPP-IV inhibitory peptide TTAGLLE from cowpea bean

425

has been shown by Molecular docking to bind to S3 pocket (one of the active site in

426

DPP-IV), indicating that interactions with the S3 pocket are an important predictors of

427

molecules that inhibit DPP-IV.43 DPP-IV has two binding pockets and one connection

428

area as follows: the S1 pocket involves residues Ser630, Asn710 and His740; the S2

429

pocket (connection area)consists of the Arg125, Glu205 and Glu206, and the S3

430

pocket consists of Tyr547, Arg358 and Phe357.45 As shown in Fig 7A-F, all of the

431

peptides had interactions with the S1, S2 or S3 pocket residues as follows: PAGPF

432

(Asn740, Ser630), Q(-17.03) KTMPGP (Tyr547, Arg358), IPQVS (Asn740, Arg125,

433

Glu206), and ELHQEEPL (Arg125, Glu206, Arg358).

434

Some phenomena found in Molecular docking studies on IPQVS and ELHQEEPL

435

were different from that in vitro experiments: DPP-IV inhibitory activity of

436

ELHQEEPL was superior to IPQVS. Fortunately, de Mejia et al. found that the

437

optimization of the space structure of peptide by Accelrys Discovery software

438

contributes to improve the minimum evaluation of the force in Molecular docking.42

439

Therefore, optimized by Accelrys Discovery Studio 4.0, each of three peptides and

440

one modified peptide has its own independent bond energy range. Among the selected 21


Page 22 of 49

Page 23 of 49


441

peptides, putative complex between sequences IPQVS and ELHQEEPL with DPP-IV

442

were presented in Fig 8A and B. The binding of the peptide IPQVS to DPP-IV was

443

the best, which was consistent with the in vitro results. Fig 8A shows the putative

444

complex between IPQVS and DPP-IV, revealing the key ionic interactions, which

445

involved its charged termini and seemed to play a predominant role. A double salt

446

bridge involving Glu205 and Glu206 formed by the N-terminus of IPQVS, which

447

matched the molecular docking result of the Diprotin A (Fig 8C).46 When the atom H

448

on the amino group is substituted by a substituent group, the degree of DPP-IV

449

binding is lowered due to steric factors and electrical properties, and the activity is

450

reduced.26 Meanwhile, the middle position of V4 (Val) and Try547 also formed a

451

double salt bridge which is similar to that of Diprotin A (Fig 8B). Because of these

452

interactions, the interaction between IPQVS and DPP-IV was increased, resulting in

453

the activity of the peptide IPQVS being the best. However, there was a great

454

difference between the peptide IPQVS and Diprotin A. For Diprotin A, in addition to

455

the force mentioned above, the carboxyl terminus stabilized two ion-pairs with

456

Asn710 and Try547, respectively. The space of the S1 pocket was narrower than that

457

of S2, but the hydrogen bond formed by the medium polar electrophilic group in the

458

peptide could not be ignored,47 which caused the activity of the peptide IPQVS to be

459

weaker than that of the Diprotin A.

460

Diprotin A can be hydrolyzed by DPP-IV into Ile and Ile-Pro, and the degradative

461

Ile-Pro continues a moderate inhibiting effect against DPP-IV.48 The peptide IPQVS

462

could hydrolyzed in a similar manner to Diprotin A due to its N-terminal amino acid 22



463

residue composition. A similar structure containing Xαα-Pro or Xaa-Pro-Yaa can bind

464

to the active center of DPP-IV competitively, and in this situation, its affinity is much

465

greater than that in a natural substrate, thereby reducing the catalytic activity of

466

DPP-IV.43,

467

peptide ELHQEEPL, which explained why IPQVS had stronger DPP-IV inhibitory

468

activity than ELHQEEPL in vitro, even though the number of effective sites with

469

DPP-IV was not dominant in the molecular docking. Peptides IPQVS and

470

ELHQEEPL might be excellent DPP-IV inhibitors as predicted by Molecular docking

471

because hydrogen bond interactions and charged amino acids showed a positive

472

correlation with DPP-IV inhibiting activity50, which is correlated with the in vitro

473

results for the DPP-IV inhibitory activity assay, demonstrating that various aspects

474

must be considered and that evaluation models should be constructed together with

475

various parameters while evaluating the DPP-IV inhibitory activity of peptides.

49

However, there is none Xaa-Pro or Xaa-Pro-Yaa at the N-terminus in

476

In conclusion, the present results are the first to report the identification and

477

quantification of DPP-IV inhibitory peptides derived from Napin. Molecular docking

478

provided a better understanding of the molecular principle of DPP-IV inhibitory

479

peptides. The goal of the evaluated DPP-IV inhibitory peptide is to block the

480

degradation of GLP-1, which regulates blood glucose levels by stimulating insulin

481

secretion. Napin-derived processing peptides can be used as a dietary supplement for

482

the prevention of Type 2 diabetes. Furthermore, the transepithelial transport

483

mechanism of the four peptides will be further studied based on Caco-2 cell line, and

484

STC-1 murine cell line will be used to investigate the expression of GLP-1 regulated 23


Page 24 of 49

Page 25 of 49

485


by them.

486 487

Abbreviations Used

488

DPP-IV, Dipeptidyl Peptidase IV;

489

PAGPF, Pro-Ala-Gly-Pro-Phe;

490

IPQVS, Ile-Pro-Gln-Val-Ser;

491

Q (-17.03) KTMPGP, Gln-Lys-Thr-Met-Pro-Gly-Pro;

492

ELHQEEPL, Glu-Leu-His-Gln-Glu-Glu-Pro-Leu;

493

GLP-1, Glucagon-like peptide-1;

494

GIP, Glucose-dependent insulin tropic polypeptide;

495

DH, Degree of Hydrolysis;

496

TIC, Total iron chromatogram;

497

M1, Modified peptide 1;

498

M2, Modified peptide 2;

499

MD, Molecular docking;

500

PDB, Protein Data Bank;

501

MOE, Molecular Operating Environment;

502

REU, Rosetta energy units;

503

Reversed-phase high-performance liquid chromatography, RP-HPLC.

504 505 506

Supporting Information Description 24



507

Method of Folin-phenol:

508

(1) Reagent preparation

509

250 μg/mL bovine serum albumin solution was waiting for used; Folin phenolic Ⅰ

510

solution was taken in a ratio of 50:1of A solution (5 g NaOH, 0.5 g

511

C4H4O6KNa·4H2O, 20 g Na2CO3 dissolved in 1000 mL deionized water) and B

512

solution (1g CuSO4 dissolved in 200 mL deionized water).

513

(2) Standard curve

514

Different amounts of standard protein solution were taken and appropriately

515

diluted, 5 mL of Folin phenolic Ⅰ solution was added and immediately mixed, then

516

placed at room temperature for 10 min. 0.5 mL of Folin phenol Ⅱ solution was

517

added, and immediately mixed, water-soluble at 55℃ for 5 min, cold water bath for

518

10 min, and then the absorbance was measured at 650 nm. A standard curve was

519

drawn based on the obtained values.

520

(3) Determination of sample protein concentration

521

The sample to be tested was appropriately diluted in deionized water so that the

522

protein concentration was 0.25-5.00 mg, and 1 mL of the sample solution was added

523

with Folin phenol I solution and II solution, and the subsequent operation steps were

524

the same as the standard curve operation. Through the absorbance of the sample to be

525

tested, the protein content was first calculated on the standard curve, and then

526

multiplied by the dilution factor, and finally the protein content of the sample was

527

determined.

528

Kjeldahl method: 25


Page 26 of 49

Page 27 of 49


529

Method used was as described in Netto, A. D. P. (2013). Samples were digested

530

using a rapid determination system that consisted of a digestion unit with a scrubber

531

and a distillation unit. Concentrated H2SO4 (2 mL) and a small amount of catalyst

532

(K2SO4 and CuSO4) were used. The digested samples were distilled after reacting with

533

10 mL of 40% w/v NaOH solution. The distillate was collected in H3BO3 solution

534

(2% w/v) and titrated using a HCl solution (0.01400 mol/L). The endpoint of the

535

titration was determined by potentiometry. The percentage of proteins was obtained

536

by multiplying the total nitrogen content (expressed in mg of nitrogen per millilitre of

537

rapeseed) by 6.25, the standard conversion factor.

538 539

Figure S1

540

Error bars and correlation coefficient of Lineweaver–Burk double reciprocal plots

541 542

Funding

543

This work was supported by the National Key Research and Development Program

544

of China (2016YFD0400201), the National Natural Science Foundation of China

545

(31871729),

546

China(2016YFD0400206-3), the Natural Science Foundation of Jiangsu Province

547

(BK20150985), the Science and Technology Development Program of Nanjing

548

(20177835), and a Project Funded by the Priority Academic Program Development of

549

Jiangsu Higher Education Institutions (PAPD).

550

Notes

the

National

Key

Research

and

26


Development

Program

of


551

The authors declare no competing financial interest.

552 553

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554

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47. García-Aparicio C, Bonache M C, De Meester I, et al. Design and discovery of a

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novel dipeptidyl-peptidase IV (CD26)-based prodrug approach. J. Med. Chem., 2006,

691

49(17): 5339-5351.

692

48.Zhang Y, Chen R, Chen X, et al. Dipeptidyl peptidase IV-inhibitory peptides

693

derived from Silver carp (Hypophthalmichthys molitrix val.) proteins. J. Agric. Food

694

Chem., 2016, 64(4): 831-839.

695

49.Lacroix I M E, Li-Chan E C Y. Dipeptidyl peptidase-IV inhibitory activity of dairy

696

protein hydrolysates. Int. Dairy J., 2012, 25(2): 97-102.

697

50.Mojica L, de Mejía E G. Optimization of enzymatic production of anti-diabetic

698

peptides from black bean (Phaseolus vulgaris L.) proteins, their characterization and

699

biological potential. Food Funct., 2016, 7(2): 713-727.

700 701

Author Information

702

First author:

703

Feiran Xu, 0000-0001-7282-658X, [email protected]

704

Corresponding Author: 33


Page 34 of 49

Page 35 of 49


705

Xingrong Ju, [email protected]

706

Lifeng Wang, 0000-0002-0443-9649, [email protected]

707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726

Figure Captions 34



727

Figure 1 SDS-PAGE of Napin (line 1), Cruciferin (line 2), and rapeseed albumin

728

protein (line 3); all of lines in the presence of 2-mercaptoethanol.

729 730

Figure 2 (A) Sephadex G-15 gel chromatogram of MW < 1 kDa fraction from Napin

731

hydrolyzates.

732

(B) DPP-IV IC50 for Napin hydrolyzates at different GX, expressed in mg of dry

733

hydrolysate per mL (mg hydrolysate/mL). Each bar represents the mean and standard

734

deviation of three determinations. Bars labeled with different letters are significantly

735

different (P < 0.05).

736

(C) Representative spectra of Napin hydrolyzates (Alcalase + Typsin) TIC (total iron

737

chromatogram).

738 739

Figure 3 (A) Preparative RP-HPLC profile of fraction G2-Rx from Napin hydrolyzate

740

G2.

741

(B) Samples were tested at a concentration of 1.00 mg/mL and data is displayed as %

742

inhibition. Mean ± SD (n = 3). Values with different letters for fractions with DPP-IV

743

inhibitory activity are significantly different at p < 0.05.

744 745

Figure 4 (A) Semi-preparative RP-HPLC profile of fraction G2-R2 from Napin

746

hydrolyzate;

747

(B) Characteristic peaks of G2-R2 analyzed by annotated chromatogram of protein

748

P17333. 35


Page 36 of 49

Page 37 of 49

749


(C) Enzymatic sequence analysis of peptide-derived Napin (P17333).

750 751

Figure 5 HPLC chromatograms identified peaks and Triple TOF MS/MS identified

752

peaks for (A) PAGPF, (B) Q (-17.03) KTMPGP, (C) IPQVS, (D) ELHQEEPL.

753 754

Figure 6 Lineweaver−Burk double-reciprocal plot for DPP-IV activity in the absence

755

and presence of Napin-derived peptides at inhibitory concentrations of 0, 50, and 100

756

mM. (A) peptide PAGPF, (B) peptide Q(-17.03)KTMPGP, (C) peptide IPQVS, (D)

757

peptide ELHQEEPL.

758 759

Figure 7 Key interactions stabilizing the putative complex between four peptides (A.

760

PAGPF, B.Q(-17.03)KTMPGP, C.IPQVS, D.ELHQEEPL and DPP-IV without

761

structural optimization. The displayed protein residues are also involved in key

762

interactions with (E) Diprotin A and (F) omarigliptin as seen in the utilized resolved

763

DPP-IV structure.

764 765

Figure 8 The binding modes of DPP IV with peptide IPQVS and Diprotin A,

766

respectively. (A), (B), and (C) represent the combination pattern of DPP-IV/IPQVS,

767

DPP-IV/ ELHQEEPL, and DPP IV/Diprotin A, respectively.

768 769

36



Page 38 of 49

Tables Table 1 DPP-IV Inhibitory Activities and Degree of Hydrolysis of Hydrolyzed Rapeseed Protein-Derived Napin Proteins Obtained by Double Enzyme Treatment. Elastase type

Hydrolysis conditions with

DH*

DPP-IV inhibition

elastase

(%)

IC50 (mg/mL) #

pH

Time

T

E:S

(h)

(°C)

(%)

Alcalase+

8.0

3.0

55

2.0

Trypsin

8.0

2.0

37

1.5

Alcalase+

8.0

3.0

55

2.0

Pepsin

2.0

2.0

37

2.0

Alcalase+

8.0

3.0

55

2.0

Flavouzyme

9.0

2.0

50

2.0

Alcalase+

8.0

3.0

55

2.0

Papain

6.5

2.0

55

0.5

*Data

3KD

15.06± 2.70a

0.68±0.09a

2.28±0.18f

>5.00

16.24± 1.55ab

1.54±0.18cd

1.90±0.22e

>5.00

20.57± 1.87e

0.89±0.55b

2.22±0.69f

3.51±0.52g

18.62± 2.62cd

1.87±0.19de

2.09±0.43ef

>5.00

Expressed As Mean ± Standard Deviation (n=3). Different Superscript

Characters Represent The Significant Difference At p200

4

IPQVS

542.3064

18.37

-1.1

PEAKS DB

52.16±5.91b

5

NIPQVS

656.3493

18.86

-2.7

PEAKS DB

>200

6

KTMPGPS

716.3527

22.09

-4.4

PEAKS DB

>200

7

HQEEPL

751.3500

23.51

-0.8

PEAKS DB

>200

8

ELHQEEPL

993.4767

23.84

-0.1

PEAKS DB

78.46±4.94b

M1

Q(-17.03)QWLH

693.3235

19.22

-0.8

PEAKS PTM

>200

M2

Q(-17.03)KTMPGP

740.3527

22.82

-1.3

PEAKS PTM

162.73±12.26f

Diprotin A

IPI

341.2896

nd

nd

nd

3.62±0.37a

*Data

Expressed As Mean ± Standard Deviation (n=3). Within The Same Column,

Different Superscript (a-f) Characters Represent The Significant Difference At p

Identification and Quantification of DPP-IV Inhibitory Peptides from

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