Insights into the Mechanism of Quercetin against BSA-Fructose

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Bioactive Constituents, Metabolites, and Functions

Insights into the Mechanism of Quercetin against BSAFructose Glycation by Spectroscopy and High Resolution Mass Spectrometry: Effect on Physicochemical Properties Lu Zhang, Yu Lu, Yun-hua Ye, Si-hang Yang, Zong-cai Tu, Juan Chen, Hui Wang, Hong-hong Wang, and Tao Yuan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06075 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 23, 2018

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Journal of Agricultural and Food Chemistry

1

Insights into the Mechanism of Quercetin against BSA-Fructose

2

Glycation

3

Spectrometry: Effect on Physicochemical Properties

4

Lu Zhang,† Yu Lu,† Yun-hua Ye,†,* Si-hang Yang,† Zong-cai Tu,†,‡ Juan Chen,† Hui Wang,†,‡

5

Hong-hong Wang,† and Tao Yuan†,§,*

by

Spectroscopy

and

High

Resolution

Mass

6 7

†National

8

Freshwater Fish High-value Utilization of Jiangxi Province, College of Life Science, Jiangxi

9

Normal University, Nanchang, Jiangxi 330022, China

R&D Center for Freshwater Fish Processing, and Engineering Research Center of

10

‡State

11

330047, China

12

§The

13

of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of

14

Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China

Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi

Key Laboratory of Plant Resources and Chemistry of Arid Zone, and State Key Laboratory

15 16

* Corresponding authors:

17

Prof. Yun-hua Ye

18

Phone: +86-791-8812-1868;

19

Fax: +86-791-8830-5938;

20

E-mail: [email protected];

21 22

Prof. Tao Yuan

23

Phone/Fax: +86-991-3690-335;

24

E-mail:

[email protected]

ACS Paragon Plus Environment


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ABSTRACT

26

Quercetin has been reported to suppress protein glycation or the formation of advanced glycation

27

end-products (AGEs), but the inhibition mechanism related to protein structure and glycation sites,

28

and the influence on physicochemical properties remain unclear. The aim of the current research

29

was to investigate the mechanism of quercetin against glycation with BSA-fructose as model by

30

spectroscopic and spectrometric techniques. Changes in physicochemical properties were

31

evaluated by antioxidant activity and emulsifying properties. The results indicated that quercetin

32

dose-dependently inhibited the glycation of BSA by attenuating the alteration of conformational

33

structure and micro-environment induced by glycation. It could also suppress the cross-linking or

34

aggregation of glycated BSA, which reflected in the decreased molecular weight determined by

35

SDS-PAGE and MALDI-TOF. Nano liquid chromatography coupled to Q-ExactiveTM tandem

36

mass spectrometry analysis revealed the mapping of 20, 23, 19 and 19 glycation sites in glycated

37

BSA with 0, 0.5, 1.5 and 3.0 mM quercetin, respectively. Quercetin changed the glycation sites of

38

BSA, but it could not reduce the number greatly. In addition, quercetin reduced the antioxidant

39

ability, increased the emulsifying properties of BSA, while negligible efficiency was observed on

40

the antioxidant activity and emulsifying activity index of glycated BSA.

41

KEYWORDS: Quercetin, Glycation, BSA-fructose, Conformational structure, Easy-nLC

42

Q-ExactiveTM

MS/MS


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ABBREVIATIONS

44

AGEs: advanced glycation end-products; BSA: bovine serum albumin; FT-IR: Fourier transform

45

infrared spectroscopy; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis;

46

MALDI-TOF: matrix assisted laser desorption ionization time-of-flight mass spectrometer;

47

Easy-nLC Q-ExactiveTM MS/MS: Easy Nano liquid chromatography system coupled to

48

Q-ExactiveTM tandem mass spectrometry; AG: aminoguanidine. Que0.5: glycated BSA with 0.5

49

mM quercetin; Que1.5: glycated BSA with 1.5 mM quercetin; Que3.0: glycated BSA with 3.0

50

mM quercetin; Que0.5B: native BSA with 0.5 mM quercetin; Que1.5B: native BSA with 1.5 mM

51

quercetin; Que3.0B: native BSA with 3.0 mM quercetin. EAI: emulsifying ability index; ES:

52

emulsifying

stability.



53

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INTRODUCTION

54

Diabetes mellitus is a global chronic metabolic disease characterized by long-term high blood

55

sugar level. It was documented that about 415 million people worldwide suffered from diabetes in

56

2015, the number was estimated to grow to 642 million by 2040 1. As a result of prolonged

57

hyperglycemia and oxidant stress in diabetes, advanced glycation end-products (AGEs) are

58

usually produced through glucose auto-oxidation, protein glycation and oxidation of protein

59

AGEs refer to a family of stable and chemically heterogeneous compounds produced through

60

glycation or Maillard reaction between the carbonyl group of reducing sugars and the free amino

61

groups of proteins, lipids and nucleic acids. It can generate oxidative stress, activate AGE

62

receptors, promote the production of inflammatory and fibrogenic growth factors and cytokines,

63

alter the structure and function of body proteins, induce microvascular damage, et al.

64

Numerous studies have indicated that the accumulation of AGEs in tissues is profoundly

65

associated with the development and progression of diabetic complications, atherosclerosis,

66

Alzheimer's, cataract and nephropathy, et al.

67

safe AGEs inhibitors from natural sources is attracting increasing attentions 7, 8.

2, 4-6.

.

2, 3

2, 4, 5.

In recent decades, exploration of effective and

68

Quercetin is one of the most abundant flavonols widely present in fruits, vegetables, cereals

69

and herbs. It has draw plenty of attentions due to its potential efficient for human health, such as

70

anti-oxidant,

71

anti-thrombic abilities 9, 10. Among which, the prevention of quercetin against protein glycation or

72

AGEs formation has been evidenced by many researchers. Wu and Yen 11 indicated that quercetin

73

exhibited significantly inhibitory activity on protein glycation in the early and middle stage, and

74

could effectively inhibit AGEs formation in bovine serum albumin (BSA)-glucose model. Wu et

anti-diabetic,

anti-inflammatory,

anti-hypertensive,


anti-arrhythmic

and

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al.12 revealed quercetin was one of the three major antiglycation agents present in guava leaf,

76

which displays over 95% inhibitory ability on albumin glycation at 100 μg/mL. In addition, it has

77

been evidenced to be a better agent than aminoguanidine in inhibiting the formation of glycation

78

products.6 The anti-glycation mechanism of quercetin proposed so far includes scavenging

79

reactive oxygen species, trapping dicarbonyl compounds, chelating metal ions.13, 14 While, to the

80

best of our knowledge, the action mechanism in terms of protein structure has not been fully

81

elucidated, influence of quercetin on the structure and glycation sites of proteins remains

82

unknown.

83

Currently, the typical techniques for structural determination of proteins are sodium dodecyl

84

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism spectroscopy (CD),

85

fourier transform infrared spectroscopy (FT-IR) and fluorescence spectra. These methods can

86

provide a global structural information of tested protein, but cannot elucidate what exactly

87

happened inside of the protein and the detailed changes in glycation sites of protein after the

88

addition of inhibitors. With the development of mass spectrometry-based techniques,

89

matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF) and

90

orbitrap tandem mass spectrometry (MS/MS) were employed as a powerful tool in precisely

91

structural characterization and unambiguous peptide mapping at a molecular level due to their

92

greatest combination of high accuracy, resolution, and sensitivity.15, 16 Up to data, MALDI-TOF

93

and Orbitrap MS/MS techniques have been successfully applied to identify the glycated peptides

94

and glycation sites of protein after glycosylated modification,17, 18 and to elucidate the mechanism

95

of physical modification on proteins glycosylation.19, 20

96

The present research was thus designed to investigate the mechanism of quercetin against BSA



97

glycation by combining spectroscopic and spectrometric techniques, and to evaluate the effect of

98

quercetin on the antioxidant ability and emulsifying properties of BSA. Changes in molecular

99

weight of BSA induced by glycation and quercetin were analyzed by SDS-PAGE and

100

MALDI-TOF. Alteration in spacial conformation and secondary structure was investigated by

101

fluorescence spectrometry and FT-IR. Effect of quercetin on the glycation sites of BSA mediated

102

by fructose was elucidated by an Easy Nano liquid chromatography system coupled to a

103

Q-ExactiveTM tandem mass spectrometry (Easy-nLC Q-ExactiveTM MS/MS). The antioxidant

104

ability was tested by DPPH·and ABTS·+ scavenging ability, the emulsifying properties was

105

analyzed by emulsifying ability index (EAI) and emulsifying stability (ES).

106

MATERIALS AND METHODS

107

Materials. Aminoguanidine (AG), BSA, trypsin, α-cyano-4-hydroxycinnamic acid (α-CHCA)

108

and C18 Cartridge were purchased from Sigma (St. Louis, MO). Urea, trisbase, DL-dithiothreitol

109

(DTT) and iodoacetamide (IAA) were from Bio-rad (CA, USA). Mass Standards Kit for the 4700

110

Proteomics Analyzer was provided by AB SCIEX (CA, USA). Formic acid, trifluoroacetic acid

111

and acetonitrile were purchased from Merck (NY, USA). Water was purified by a Milli-Q water

112

purification system (Milford, MA). All reagents used for HPLC analysis were HPLC grade. All

113

other reagents used were of analytical grade and purchased from Solarbio Chemical Co. (Shanghai,

114

China).

115

Preparation of Glycated BSA. Fructose (0.625 M), BSA (25 mg/mL) and aminoguanidine (1.5

116

mM) solution were all prepared with 200 mM, pH 7.4 PBS. Then, 3.0 mL of BSA was incubated

117

with 3.0 mL of fructose and 50 μL of quercetin at various concentrations (0.5, 1.0, 1.5, 2 and 3.0

118

mM). The blank group and control group without fructose or quercetin were prepared in parallel to


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analyze or exclude the effect of quercetin. After incubation at 37 oC for 7 d, the reaction solutions

120

were kept at 4 0C for further analysis.

121

Inhibition on AGEs formation. The inhibitory activity of quercetin on AGEs formation 13

122

was evaluated according to the method of Bhuiyan et al.

with minor modifications. All sample

123

solutions were 10-fold dilution before the measurement of fluorescence intensity at an excitation

124

and emission wavelength of 350 nm and 425 nm, respectively. The data were recorded using a

125

Hitachi F-7000 fluorescence spectrometer (Tokyo, Japan) .

126

Intrinsic Fluorescence. Effect of quercetin on the intrinsic emission fluorescence spectra

127

of glycated and native BSA was performed on a Hitachi F-7000 fluorescence spectrophotometer

128

(Hitachi, Tokyo, Japan). The sample solutions were diluted with 0.2 M, pH 7.4 PBS for ten times,

129

and then submitted to spectrum analysis at an excitation wavelength of 280 nm. The emission

130

spectra were recorded from 300 to 400 nm at a constant slit of 5 nm for both excitation and

131

emission.

132

FT-IR Spectroscopy Analysis. The BSA solutions were ultra-filtrated with

133

Amicon-Ultra-15 centrifugal filter (Millipore, Bedford, MA) (10 kDa cutoff) by using a high

134

speed refrigerated centrifuge before using. The unbound quercetin, fructose and salt were removed

135

by centrifuging at 10,000 rpm for 10 min, followed by washing with ultra-pure water for three

136

times. The remained BSA solution in the upper part of the tube was washed out with ultra-pure

137

water and freeze-dried for further analysis.

138

The FT-IR analysis of all purified BSA samples were performed on an FT-IR spectrometer

139

(Spectrum One, Perkin Elmer, USA) equipped with a DTGS detector. The dry samples were



140

grinded with KBr thoroughly and then compressed to form slices. Background noise was corrected

141

with pure KBr data. The spectra were recorded in the range of 400 ~ 4000 cm-1 for 32 scans at a

142

resolution of 4 cm-1.

143

SDS-PAGE Analysis. The SDS-PAGE was carried out on a Bio-rad Mini protean Tetra

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MP4 electrophoresis (Bio-Rad Mini-Protean System) using 12% separating gel and 5% stacking

145

gel.21 Samples (0.5 mg/mL) were mixed with loading buffer (No. P1016) at a ratio of 3:1 (v/v) and

146

boiled for 5 min. After centrifugation at 10,000 g for 1 min, 10 μL of each sample was loaded on

147

the gel lanes for electrophoresis. After the electrophoresis, the gel sheets were stained with 0.05%

148

Coomassie Blue R-250, and destained with 40 % (v/v) methanol containing 10 % (v/v) acetic acid.

149

MALDI-TOF Mass Spectrometry Analysis. Molecular weight analysis of

150

ultra-filtrated BSA was conducted on a 4800 Plus MALDI TOF/TOFTM Analyzer (AB SCIEX,

151

CA, USA) under positive ion mode.22 CHCA was used as the matrix, 4700 Proteomics Analyzer

152

Calibration Mixture 1 was used as calibration standard with mass error less than 0.5 Da. All

153

spectra were acquired in the mass range of 20000-200000 m/z, each spectrum was collected with

154

500 laser shots.

155

Liquid Chromatography and Mass Spectrometry Analysis

156

Digestion of samples. The freeze-dried BSA were suspended in 200 μL SDT buffer (4%

157

SDS, 100 mM Tris-HCl, 1 mM DTT, pH 7.6) and boiled for 5 min. Then, 50 μL of sample

158

solution was added to a 10 kD ultra-filtration centrifuge tube (Sartorius, Germany) containing 200

159

μL of UA buffer (8 M Urea, 150 mM Tris-HCl, pH 8.0) and centrifuged (Eppendorf5430R,

160

Germany) at 14000 g for 15 min. After another centrifugation at 14000 g for 15 min with 200 μL


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of UA buffer, the filtrate was removed, 100 μL of IAA (50 mM IAA in UA buffer) was added and

162

shaken thoroughly. The mixture was allowed to incubate at room temperature for 30 min,

163

followed by centrifugation at 14000 g for 10 min. The BSA solution in the upper part of the tube

164

was then successively washed with 100 μL of UA buffer and 100 μL of 25 mM NH4HCO3

165

solution by centrifuging at 14000 rpm for 10 min, each washing was performed twice. Finally, 40

166

μL of trypsin buffer (2 μg trypsin in 40 μL 25 mM NH4HCO3) was added to hydrolyze the BSA

167

samples at 37 °C for 16 h. The proteolytic peptides were collected by centrifuging at 14000 g for

168

10 min and used for peptide mapping.

169

MS/MS analysis. An Easy-nLC1000 system couple to a Q-ExactiveTM tandem mass

170

spectrometer (Thermo Fisher) was used for the analysis of proteolytic peptides. Two microliters of

171

digested samples were injected onto a Thermo scientific EASY column (75 μm × 100 mm, 3

172

μm-C18). After desalting for 5 min with 4% B (0.1% FA in 84% acetonitrile), the peptides were

173

eluted with solvent A (0.1% FA in H2O) and solvent B at a gradient of 4%-50% B for 50 min,

174

50%-100% B for 4 min and 100% for 6 min. The flow rate was 300 nL/min. The effluent was

175

directly infused into a Q-ExactiveTM mass spectrometry for peptide mapping under positive mode.

176

The MS and MS/MS spectra of peptides were collected in full scan mode. High energy capture

177

dissociation (HCD) fragmentation mode was used to acquire the fragment ions of each peptide.

178

Antioxidant activities. The DPPH· and ABTS·+ scavenging ability of glycated and native BSA

179

was measured according to the method of Gu et al.23 and Liu et al.24, respectively, with some

180

modifications. An aliquot of 50 μL of 4-fold diluted samples were reacted with 150 μL of 0.15

181

mM DPPH solution in ethanol for 30 min in darkness. Then, absorbance As at 510 nm were

182

recorded with a micro-plate reader. Control group (Ac) and blank group (Ab) were prepared in



183 184

parallel. Percentage inhibition was calculated as follows: Inhibition (%) 

Ac  ( As  Ab) 100% Ac

Eq.(1)

185

In case of ABTS·+ scavenging ability, ABTS·+ solution was diluted with 5.0 mM, pH 7.4 PBS

186

to an absorbance of 0.70 ± 0.20 at 734 nm before use. Then, 50 μL of 8-fold diluted samples were

187

reacted with 150 μL ABTS·+ solution in 96-well micro-plate at 25 oC for 6 min, absorbance (As)

188

at 734 nm was read by using a micro-plate reader. Control group (Ac) and blank group (Ab) were

189

prepared in parallel. The percentage inhibition was calculated according to Eq.(1).

190 191

Emulsifying Properties. Effect of glycation and quercetin on the EAI and ES of BSA was analyzed using the method applied by Liu et al.21 The EAI and ES were calculated as follows: 2  2.303  A0 F M

192

EAI (m 2 /g ) 

193

ES (%)  A 0  t/ (A 0  A10 )

Eq.(2)

Eq.(3)

194

where F is the oil volume fraction (0.25), M is the weight of BSA (g), A0 and A10 is the

195

absorbance measured immediately and 10 min after emulsion formation, respectively, △t =10 min.

196

Data Analysis. All experiments were performed in triplicate, and the results were presented

197

as mean value ± standard deviation (SD). Statistically significant difference was determined by

198

one-way analysis of variance (ANOVA) using SPSS 13.0. A value of p < 0.05 represents

199

significant difference. The MS and MSMS data were processed with Q-Exactive 2.0 software

200

(Thermo, MA, USA), the peptides and glycation sites were identified by submitting the MS and

201

MS/MS spectra to Mascot server through Proteome Discoverer software. The search parameters

202

were set as: enzyme: trypsin; missed cleavage: 2; fixed modification: carbamidomethyl of cysteine

203

(Cys); variable modification: oxidation of methionine (Met) and glycation of lysine (Lys),

204

arginine (Arg) and N-terminal; peptide tolerance: ± 10 ppm; MS/MS tolerance: ± 0.2 Da.


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RESULTS AND DISCUSSION

206

Inhibition on AGEs Formation. In this research, the inhibitory ability on AGEs

207

formation was used to reflect the ability of quercetin against BSA glycation, which was measured

208

by recording the fluorescence intensity at 350 nm of excitation and 425 nm of emission

209

wavelength. Lower fluorescence intensity implies stronger inhibitory capacity. To simplify the

210

expression of different glycated BSA, BSA-fructose, Que0.5, Que1.5 and Que3.0 was applied to

211

represent the BSA glycated with the addition of 0, 0.5, 1.5 and 3.0 mM of quercetin, respectively.

212

As shown in Figure 1, quercetin dose-dependently inhibited the formation of fluorescent AGEs,

213

the fluorescence intensity of glycated BSA declined from 247.77 to 75.40 when the concentration

214

of quercetin was increased from 0 to 3.0 mM. While, AG showed negligible inhibition on AGEs

215

with the fluorescence intensity of 243.73 at 3.0 mM, suggesting strong inhibition of quercetin on

216

AGEs formation. A strong and dose-dependent inhibition of quercetin on fluorescent AGEs was

217

also observed by Alam et al.25 in human serum albumin-glucose model. The excellent inhibition of

218

quercetin on AGEs could be partially due to its strong antioxidant activity, previously published

219

literatures evidenced that quercetin can inhibit AGEs formation by scavenging the reactive oxygen

220

species and trapping the dicarbonyl compounds produced during the early and middle stage of

221

glycation.13, 14 Positive correlation between anti-glycation potential and antioxidant properties of

222

compounds or extract has been observed by many researches 26, 27. At the concentration of 0.5, 1.0,

223

1.5, 2.0 and 3.0 mM, the percentage inhibition of quercetin on AGEs formation was 7.06, 29.34,

224

41.33, 59.32 and 69.57%, respectively. Thus, native and glycated BSA treated with 0, 0.5, 1.5 and

225

3.0 mM of quercetin were selected for future structural analysis and glycated peptides

226

identification.



227

Intrinsic Fluorescence Analysis. Fluorescence quenching is the decrease of quantum

228

yield of fluorescence when the chromophores interact with quenching agents. The intrinsic

229

fluorescence spectrum is mainly depend on the polarity of the environment of Trp residues, and

230

can reflect the conformation changes of proteins in receptor-ligand interaction.22, 28

231

As shown in Figure 2, the native BSA has a strong fluorescence-emission peak at 343.2 nm

232

after being excited at 280 nm, but it decreased dramatically after being glycated with fructose for

233

7 days, with the relative fluorescence intensity (RFI) reduced from 9682 to 3176, and the λem was

234

shifted from 343.2 to 345.2 nm. BSA has two tryptophan moieties that possess intrinsic

235

fluorescence including Trp 134 and Trp 212. Trp 134 locating in subdomains I is more exposed to

236

hydrophilic environment, whereas Trp 212 locating in subdomains II is deeply buried in the

237

hydrophobic binding pocket.29 The reduced RFI and red shift of λmax indicated that glycation

238

altered the conformational structure of BSA and changed the micro-environment around

239

fluorescence chromophore.30, 31 Significant decrease in intrinsic fluorescence intensity and a red

240

shift of λmax in fluorescence spectra of protein induced by glycation were also reported by other

241

researchers.32,

242

quenching of BSA induced by glycation was detected, the RFI of glycated BSA elevated gradually

243

with increasing quercetin concentration, the λmax was also returned from 345.2 nm to 342.8 nm,

244

implying a preventative effect of quercetin on the conformation and micro-enviromental alteration

245

of BSA induced by glycation reaction.34 Alam et al.

246

inhibited the structural loss of glycated BSA.

33

Upon addition of quercetin, a dose-dependent alleviation on the fluorescence

25

indicated that quercetin dose-dependently

247

While in case of native BSA, quercetin showed a dose-dependent quenching effect on the

248

tryptophan fluorescence. The RFI declined from 9384 to 8370 when the concentration of quercetin


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249

was increased from 0.5 mM to 3.0 mM, but no apparent red or blue shit of λem was observed.

250

Quercetin is a hydrophobic molecule that can penetrate into the hydrophobic loop of BSA, and

251

expose the fluorescence chromophore of BSA to a more hydrophobic environment, leading to

252

declined RFI, which is agreement with the results found by Papadopoulou et al.28. Many other

253

compounds, such as fosfomycin, rutin, methimazole and 6n-propyl-2-thiouracil, were also

254

reported to decrease the fluorescence of BSA.28, 29, 35

255

FT-IR Analysis. The FT-IR spectra of native and glycated BSA with and without quercetin

256

were measured to evaluate the possible interaction of fructose and quercetin with BSA. Figure 3

257

presents the FT-IR spectra of native and glycated BSA, region of 3450-3300 cm-1 refers to OH and

258

-NH2 stretching vibrations

259

band, 1600-1500 cm-1 represents C=N stretching and NH bending vibrations from amide II band 37;

260

region of 1100 - 1140 cm-1 refers to the stretching of C-C and C-O and the bending vibration of C-H

261

bond 38.

36;

1700-1600 cm-1 represents C=O stretching vibration from amide I

262

Glycation and conjugation with quercetin might change the IR spectrum of BSA as a result of

263

the consumption of some functional groups and the appearance of others. In this research, distinct

264

spectral shifting was only observed on regions of 3400-3300 cm-1 and 1100-1050 cm-1 after

265

glycation or the addition of quercetin. The maximum absorption peak at 3461.6 cm-1 for native

266

BSA was shifted to 3430.7, 3388.3, 3403.7 and 3380.1 for glycated BSA, Que0.5, Que1.5 and

267

Que3.0, respectively. While, the values were 3415.3, 3419.2 and 3419.2, for Que0.5B, Que1.5B

268

and Que3.0B, respectively. It indicated that both glycation and quercetin can lead to the blue shift

269

of maximum absorption at 3450-3300 cm-1. The absorption peak at 1085.7 cm-1 of native BSA

270

was shifted to 1062.6 cm-1 after glycation, however, no remarkable red or blue shifts was observed



271

on native BSA or glycated BSA with different concentrations of quercetin, suggesting that

272

quercetin has no influence on this band of native and glycated BSA. But a notable enhancement in

273

the intensity of region 1100 - 1140 cm-1 was detected on glycated BSA, revealing that fructose

274

was attached to BSA by glycation reaction.23 On the other hand, no other significant changes were

275

observed, except for the blue shift of absorption peak at 3461.6 and the broaden of region

276

3200-3600 cm-1 when quercetin was added to the native or glycated BSA. It herein could be

277

speculated that quercetin could alter the conformational structure of BSA, but no covalent bonds

278

formed between them. Previous studies indicated that quercetin bind to human serum albumin

279

predominantly through hydrophobic contacts within the hydrophobic core,25 hydrogen bonding,

280

ionic and hydrophobic interaction are equally important driving forces for BSA-quercetin

281

association.28

282

Changes in Molecular Weight. The native and glycated BSA with the addition of 0, 0.5,

283

1.5 and 3.0 mM of quercetin were applied to SDS-PAGE analysis to elucidate the influence of

284

glycation and quercetin on the molecular weight of BSA. As shown in Figure 4A, native BSA

285

gave an obvious protein band at about 66.2 kDa, which is consistent with the molecular weight of

286

BSA (66.4 kDa), the presence of quercetin has no effect on the molecular weight of BSA as

287

indicated by lanes Que0.5B, Que1.5B and Que3.0B. While, a pronounced protein band at a

288

molecular weight nearly 97.4 kDa corresponding to the tripolymer of BSA emerged on glycated

289

BSA, and the color for the polydispersed bands at the top of the separating gel with molecular

290

weight above 100 kDa was also much deeper than that of native BSA, suggesting the formation of

291

high molecular weight conjugate or the cross-linking of BSA induced by glycation. However,

292

after the addition of quercetin, the bands near and above 97.4 kDa faded, which is especially


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293

obvious for glycated BSA treated by 3.0 mM of quercetin. Thus, it can be concluded that

294

quercetin could suppress the cross-linking of BSA, which may be one of the mechanism of

295

quercetin against AGEs formation on BSA-fructose model. Previous researches also indicated that

296

polyphenols could alleviate the formation of AGEs by inhibiting the aggregation of glycated

297

protein.11, 26

298

To elucidate the alteration of molecular weight more accurately, MALDI-TOF mass

299

spectrometry analysis was employed. As shown in Figure 4B, predominant molecular weight

300

distribution for native BSA was at about 66.61kDa, which drastically increased to 67.50 kDa after

301

glycation. However, the changes were less pronounced in all glycated BSA treated by quercetin,

302

with the average molecular weight of 67.14, 67.10 and 66.88 kDa, respectively, when 0.5, 1.5 and

303

3.0 mM quercetin was added. Similar changes were also observed around 80.45 and 33.37 kDa.

304

While, for native BSA, no obvious changes were observed on the molecular weight when different

305

concentrations of quercetin were added (Fig.1S in Supplementary materials), thus, it can be

306

concluded that quercetin will not lead to the aggregation of BSA, which is consistent with the

307

results found by SDS-PAGE analysis. Above results suggested that the molecular weight of BSA

308

increased due to the attachment of fructose induced by glycation, quercetin showed a protective

309

effect against the glycation between BSA and fructose, and it had little influence on the molecular

310

of BSA.

311

Identification of Glycated Peptides and Glycation Sites. To better elucidate the

312

inhibition mechanism of quercetin on AGEs formation in BSA-fructose model, the glycation sites

313

of glycated BSA with and without quercetin were monitored by nLC Q-ExactiveTM MS/MS. It has

314

been proved that hydrogen bonding, ionic and hydrophobic interaction are the major driving



Page 16 of 36

315

forces for BSA-quercetin association,28 and quercetin bind to human serum albumin

316

predominantly through hydrophobic contacts within the hydrophobic core.25 The FI-TR and

317

MALDI-TOF analysis performed in this research also suggested that no covalent bond or

318

chemical reaction occurred between quercetin and BSA. Therefore, the glycated peptides and

319

glycation sites were mapped on the base of that no quercetin linked to the proteolytic peptides

320

after the digestion of glycated BSA with trypsin.

321

In theory, all unglycated peptides and the corresponding glycated peptides coexist in the sample

322

solution and are eluted at the similar retention time. Therefore, the glycated forms of the peptides

323

can be detected from the mass shift induced by glycation. If a peptide was glycated by a fructose,

324

the corresponding m/z peaks with charges of 1, 2, 3, 4 and 5 will display a mass shift of 162.0528,

325

81.0264, 54.0176, 40.5132 and 32.4106, respectively. The mass spectra of representative glycated

326

peptides of BSA after trypsin digestion are show in Figure 5. The m/z peak at 1244.05802+ was

327

identified as the unglycated peptides 184-204 (YNGVFQECCQAEDKGACLLPK) by matching

328

the determined HCD MS/MS spectrum with that recorded in database (Mascot and Protein

329

Prospector), its glycated form (1325.08502+) displayed an m/z difference of 81.0270, revealing

330

that the peptide was glycated by one molecule of fructose. Similarly, the detected m/z peaks of

331

unglycated

332

(VHKECCHGDLLECA DDRADLAK) and 524-544 (AFDEKLFTFHADICTLPDTEK) were at

333

1103.49533+, 653.79454+ and 833.40153+, respectively, the corresponding peaks with m/z values at

334

1157.51273+, 694.30864+, 887.41863+ were simultaneously found with individual increased mass

335

shift in m/z of 54.0174, 40.5141 and 54.0171, indicated that these peptides were all singly

336

glycated. Totally, 21, 24, 20 and 20 glycated peptides were mapped from BSA-fructose, Que0.5,

peptides

569-587

(TVMENFVAFVDKCCAADDK),


264-285

Page 17 of 36


337

Que1.5 and Que3.0, respectively, and the results were listed in Table 1. The number of glycation

338

sites was much more than that reported in previous research,20 which could be resulted from the

339

much longer glycation time (7 d) and different hydrolytic enzyme applied in this research.

340

In current research, HCD MS/MS and neutral loss detection were conducted to determine the

341

accurate peptide sequences and glycation sites. It is well-known that lysine (Lys) residues,

342

arginine (Arg) residues and N-terminal are the major potential sites of glycation,20 and the last

343

amino acid residue of a identified peptide can be excluded as a glycation site since the tryptic

344

truncation sites can only be those not glycated.17 The glycated sites were unambiguously

345

confirmed by matching the determined MS/MS fragment ions (either the b or y ion series) with the

346

theoretical fragmentation pattern recorded in ProteinProspector and Mascot. The HCD MS/MS

347

spectra of the selected glycated peptides 89-105, 401-420 and 372-386 of glycated BSA were

348

given in Figure 6. In terms of peptide 89-105 (487.26034+, SLHTLFGDELCKVASLR), only one

349

possible glycated site is available. Figure 6A presents the MS/MS spectrum of its singly glycated

350

form with m/z of 527.77314+, all the detected y ions (y1, y3 ~ y12), b ions (b2 ~ b6 and b8 ~ b9)

351

as well as the ions resulted from further neutral loss of H2O (18 Da) and NH3 (17 Da) matched

352

well

353

89SLHTLFGDELCK(Fru)VASLR105,

354

spectrum of the singly glycated peptide with m/z of 839.73573+ also produced a series of y ions

355

(y1 ~ y10), b ions (b2 ~ b10), along with the ions caused by further dehydration and deamination

356

of y ions (e.g. y6, y7, y10-y12) and b ions (e.g. b7 and b8) (Figure 6B), which clearly confirmed

357

the peptide sequence of

358

K411. Similarly, K374 was also identified as the glycated site by fragmenting the peptide

with

the

theoretical

fragmentation

pattern

of

glycated

peptide

confirming that the glycation occurred at K100. The MS/MS

401HLVDEPQNLIK(Fru)QNCDQFEK420


with the glycation occurred at


359

372LAK(Fru)EYEATLEECCA

K386 with m/z of 988.93882+ as shown in Figure 6C.

360

The sites observed in glycated BSA with the presence of various concentrations of quercetin are

361

shown in Table 1. It can be seen that the addition of quercetin could not change the number of

362

glycated sites greatly but alter the site, and the e-amino groups of lysine residues are the highly

363

reactive group, no glycated site was found on the guanidine groups of the arginine residues and

364

the a-amino group of N-terminus. In this research, 20, 23, 19 and 19 glycation sites were detected

365

in BSA-frutose, Que0.5, Que1.5 and Que3.0, respectively. The sites observed in BSA-fructose

366

were K28, K36, K100, K117, K130, K140, K188, K197, K266, K299, K304, K317, K386, K411,

367

K463, K498, K528, K559/561, K580 and K587. After the addition of quercetin, glycation sites

368

K117 and K386 disappeared, while K346 and K523 appeared, in addition, sites K130, K197,

369

K304, K411 and K463 were not glycated when the concentration of quercetin was at 3.0 mM.

370

What’s more, sites K130 and K285 were only observed in Que1.5, while sites K245 and K399

371

were only observed in Que3.0. Sites K401, K542 and K568 were found in Que0.5 and Que3.0, but

372

it was miss in BSA-fructose and Que1.5. Therefore, the alteration of glycation sites occurred

373

mainly in domain II (185 - 377) and domain III (378 - 583) of BSA.

374

The irregular alteration of glycation sites could be due to the changes in conformational

375

structure and micro-environment around lysine induced by quercetin. It has been evidenced that

376

the conformational structure of a protein is closely related to its glycation activity, an unfolding

377

conformation is more liable to undergo glycation,19, 22 and the presence of acidic, basic, negatively

378

and positively charged amino acids residues can promote the glycation of vicinal lysine.17, 39 The

379

fluorescence and SDS-PAGE analysis performed in this research revealed that quercetin can

380

individually prevent the unfolding of molecular conformation (Figure 2) and aggregation (Figure


Page 18 of 36

Page 19 of 36


381

4) of BSA induced by glycation reaction. Ma et al.34 provided that glucitol-core containing

382

gallotannins and maple syrup extract can stabilize the secondary structure and attenuate

383

conformational changes of BSA induced by glycation. In addition, quercetin can form hydrogen

384

bonds with the residues of Phe198, Ser384 and Pro385 of BSA, and it interact with BSA mainly

385

through hydrogen bonding, ionic and hydrophobic interaction,28 which may greatly alter the

386

mico-enviroment of Lys130, 197, 245, 346, 386, 399 and 441.

387

Antioxidant Activity. Effect of glycation and quercetin on the antioxidant ability of BSA were

388

given in Figure 7A and Figure 7B. It can been seen that glycation with fructose can slightly

389

improve the DPPH· and ABTS·+ scavenging ability of native BSA, which could be due to the

390

formation of antioxidant glycation products, such as melanoidins, reductone compounds, et al..23,

391

40

392

reduced glycation reaction induced by quercetin. Previous researches proved that glycation could

393

enhance the antioxidant ability of protein, and the activity is positively correlated to the glycation

394

degree.23, 24, 40 While, the activity of native BSA were dramatically reduced when 0.5 ~ 3.0 mM of

395

quercetin was added, the percentage inhibition was decreased from 60.48 to 13.95 ~ 21.88 for

396

DPPH·, and from 72.90 to 12.48 ~ 18.23 for ABTS·+. Proteins act as antioxidant by donating

397

hydrogen to stabilize the free radical, the hydrophobic amino acids Trp, His, Pro, and Tyr can

398

serve as hydrogen donors.19 Fluorescence analysis indicated that quercetin can quench the

399

fluorescence of BSA by interacting with Tyr and Try, and it interacts with BSA mainly through

400

hydrogen bonding, ionic and hydrophobic interaction,28 which can greatly decrease the hydrogen

401

donation ability of BSA, resulting to reduced radical scavenging ability.

402

The alleviated radical scavenging ability of Que 0.5, Que 1.5 and Que 3.0 could be owe to the

Emulsifying Property. The emulsifying ability index (EAI) and emulsifying stability index



403

(ESI) of glycated and native BSA with various concentrations of quercetin were described in

404

Figure 7C and Figure7D, respectively. Glycation can increase the EAI and ES of native BSA,

405

which is consistent with previous reports.21, 41 Interesting, quercetin could significantly improve

406

the ES of native and glycated BSA, and exhibited an obviously does-dependent relationship.

407

Meanwhile, the EAI of native BSA was also increased from 394.03 to 433.66 ~ 455.44 when 0.5~

408

3.0 mM of quercetin was mixed. These may be attributed to the increased hydrophobicity of

409

BSA-quercetin system, since quercetin is hydrophobic molecule and it bind to BSA predominantly

410

through hydrophobic contacts within the hydrophobic core.28, 42

411

In conclusion, quercetin can effectively inhibit the glycation of BSA in a dose-dependent

412

manner, and glycation reaction will induce the conformation unfolding and aggregation of BSA,

413

as well as the increase in molecular weight, while the association between quercetin and BSA

414

attenuates these changes. Addition of quercetin can alter the glycation sites, reduce the antioxidant

415

ability, increase the emulsifying properties of native BSA, but it cannot reduce the number of

416

glycation sites significantly. What’s more, quercetin possesses negligible efficiency on the

417

antioxidant activity and EAI of glycated BSA, but it enhances the ES significantly. These findings

418

provide a better understanding of the mechanism of quercetin against protein glycaton, along with

419

its effect on the antioxidant and emulsifying properties of native and glycated BSA.

420

ACKNOWLEDGEMENTS

421

This work was financially supported by the National Natural Science Foundation of China

422

(3186100750), the earmarked fund for China Agriculture Research System (CARS-45), and

423

Construction of superior scientific and technological innovation team of Jiangxi Province


Page 20 of 36

Page 21 of 36


424

(20171BCB24004).

425

Notes

426

The authors declare no competing financial interest.

427

REFERENCES

428

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429

Countries and Underserved Communities, Dagogo-Jack, S., Ed. Springer International Publishing:

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Cham, 2017; pp 1-5.

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432

(3) Lalla, E.; Lamster, I. B.; Stern, D. M.; Schmidt, A. M., Receptor for advanced glycation end

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436

Skibsted, L. H.; Dragsted, L. O., Advanced glycation endproducts in food and their effects on health.

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antioxidant potential and rat intestinal α-glucosidases inhibitory activities of quercetin, rutin, and

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isoquercetin. International Journal of Applied Research in Natural Products 2010, 2, 52-60.

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advanced glycation endproducts. Journal of Agricultural and Food Chemistry 2005, 53, 3167-3173.

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oxygen species. Bioscience, Biotechnology, and Biochemistry 2017, 81, 882-890.

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by trapping methylglyoxal and glyoxal. Journal of Agricultural and Food Chemistry 2014, 62,

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Biotechnology 2004, 26, 147-163.

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maillard reaction after disruption of the disulfide bridge evaluated by liquid chromatography and high

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resolution mass spectrometry. Journal of Agricultural and Food Chemistry 2013, 61, 2253-2262.

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conventionally and microwave-heated ovalbumin by high resolution mass spectrometry. Food

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Chemistry 2013, 141, 985-991.

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(19) Zhang, Q.; Tu, Z.; Wang, H.; Huang, X.; Shi, Y.; Sha, X.; Xiao, H., Improved glycation after

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ultrasonic pretreatment revealed by high-performance liquid chromatography–linear ion trap/orbitrap

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high-resolution mass spectrometry. Journal of Agricultural and Food Chemistry 2014, 62, 2522-2530.

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promoted by dynamic high pressure microfluidisation pretreatment revealed by high resolution mass

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spectrometry. Food Chemistry 2013, 141, 3250-3259.

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(21) Liu, J.; Tu, Z.-c.; Zhang, L.; Wang, H.; Sha, X.-m.; Shao, Y.-h., Influence of ultrasonication prior

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to glycation on the physicochemical properties of Bovine serum albumin–galactose conjugates. Food

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Science and Technology Research 2018, 24, 35-44.

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(22) Zhang, Q.; Tu, Z.; Wang, H.; Huang, X.; Sha, X.; Xiao, H., Structural changes of ultrasonicated

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bovine serum albumin revealed by hydrogen–deuterium exchange and mass spectrometry. Analytical

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and Bioanalytical Chemistry 2014, 406, 7243-7251.

487

(23) Gu, F.-L.; Kim, J. M.; Abbas, S.; Zhang, X.-M.; Xia, S.-Q.; Chen, Z.-X., Structure and antioxidant

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activity of high molecular weight Maillard reaction products from casein–glucose. Food Chemistry



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2010, 120, 505-511.

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(24) Liu, J.; Tu, Z.; Shao, Y.-h.; Wang, H.; Liu, G.-x.; Sha, X.-m.; Zhang, L.; Yang, P., Improved

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antioxidant activity and glycation of α-Lactalbumin after ultrasonic pretreatment revealed by

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High-Resolution Mass Spectrometry. Journal of Agricultural and Food Chemistry 2017, 65,

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10317-10324.

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(25) Alam, M. M.; Ahmad, I.; Naseem, I., Inhibitory effect of quercetin in the formation of advance

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glycation end products of human serum albumin: An in vitro and molecular interaction study.

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International Journal of Biological Macromolecules 2015, 79, 336-343.

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(26) Yamaguchi, F.; Ariga, T.; Yoshimura, Y.; Nakazawa, H., Antioxidative and anti-glycation activity

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of garcinol from Garcinia indica fruit rind. Journal of Agricultural and Food Chemistry 2000, 48,

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180-185.

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(27) Liu, W.; Wei, Z.; Ma, H.; Cai, A.; Liu, Y.; Sun, J.; DaSilva, N. A.; Johnson, S. L.; Kirschenbaum,

501

L. J.; Cho, B. P.; Dain, J. A.; Rowley, D. C.; Shaikh, Z. A.; Seeram, N. P., Anti-glycation and

502

anti-oxidative effects of a phenolic-enriched maple syrup extract and its protective effects on normal

503

human colon cells. Food & Function 2017, 8, 757-766.

504

(28) Papadopoulou, A.; Green, R. J.; Frazier, R. A., Interaction of flavonoids with bovine serum

505

albumin: A fluorescence quenching study. Journal of Agricultural and Food Chemistry 2005, 53,

506

158-163.

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(29) Sułkowska, A., Interaction of drugs with bovine and human serum albumin. Journal of Molecular

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Structure 2002, 614, 227-232.

509

(30) Huang, X.; Tu, Z.; Wang, H.; Zhang, Q.; Chen, Y.; Shi, Y.; Xiao, H., Probing the conformational

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changes of ovalbumin after glycation using HDX-MS. Food Chemistry 2015, 166, 62-67.


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Alteration of human serum albumin tertiary structure induced by glycation. Spectroscopic study.

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2016, 153, 560-565.

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(32) Yang, W.; Tu, Z.; Wang, H.; Zhang, L.; Xu, S.; Niu, C.; Yao, H.; Kaltashov, I. A., Mechanism of

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reduction in IgG and IgE binding of β-lactoglobulin induced by ultrasound pretreatment combined with

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dry-state

517

Journal of Agricultural and Food Chemistry 2017, 65, 8018-8027.

518

(33) Chen, Y.; Tu, Z.; Wang, H.; Zhang, L.; Sha, X.; Pang, J.; Yang, P.; Liu, G.; Yang, W., Glycation

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of β-lactoglobulin under dynamic high pressure microfluidization treatment: Effects on IgE-binding

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capacity and conformation. Food Research International 2016, 89, 882-888.

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(34) Ma, H.; Liu, W.; Frost, L.; Kirschenbaum, L. J.; Dain, J. A.; Seeram, N. P., Glucitol-core

522

containing gallotannins inhibit the formation of advanced glycation end-products mediated by their

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antioxidant potential. Food & Function 2016, 7, 2213-2222.

524

(35) Meti, M. D.; Nandibewoor, S. T.; Joshi, S. D.; More, U. A.; Chimatadar, S. A.,

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Multi-spectroscopic investigation of the binding interaction of fosfomycin with bovine serum albumin.

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Journal of Pharmaceutical Analysis 2015, 5, 249-255.

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(36) Zhang, H.; Zhang, Y.; Bao, E.; Zhao, Y., Preparation, characterization and toxicology properties

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of α- and β-chitosan Maillard reaction products nanoparticles. International Journal of Biological

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Macromolecules 2016, 89, 287-296.

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(37) Liu, G.-x.; Tu, Z.-c.; Wang, H.; Zhang, L.; Huang, T.; Ma, D., Monitoring of the functional

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properties and unfolding change of Ovalbumin after DHPM treatment by HDX and FTICR MS:

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Functionality and unfolding of Oval after DHPM by HDX and FTICR MS. Food Chemistry 2017, 227,

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(38) Cardoso, J. C.; Albuquerque, R. L. C.; Padilha, F. F.; Bittencourt, F. O.; de Freitas, O.; Nunes, P.

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S.; Pereira, N. L.; Fonseca, M. J. V.; Araújo, A. A. S., Effect of the Maillard reaction on properties of

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casein and casein films. Journal of Thermal Analysis and Calorimetry 2011, 104, 249-254.

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(39) Shilton, B. H.; Walton, D. J., Sites of glycation of human and horse liver alcohol dehydrogenase in

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vivo. Journal of Biological Chemistry 1991, 266, 5587-5592.

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(40) Liu, Q.; Kong, B.; Han, J.; Sun, C.; Li, P., Structure and antioxidant activity of whey protein

540

isolate conjugated with glucose via the Maillard reaction under dry-heating conditions. Food

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structure 2014, 145-154.

542

(41) Kim, H. J.; Choi, S. J.; Shin, W.-S.; Moon, T. W., Emulsifying properties of Bovine serum

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albumin−galactomannan conjugates. Journal of Agricultural and Food Chemistry 2003, 51,

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structure–function approach. Food Chemistry 2013, 141, 975-984.


Page 26 of 36

Page 27 of 36


548

Figure Captions:

549

Figure 1. Effect of quercetin on the fluorescence intensity of glycated BSA at an excitation and

550

emission wavelength of 350 nm and 425 nm, respectively.

551

Figure 2. Intrinsic fluorescence spectra (A) and maximum fluorescence intensity (B) of native and

552

glycated BSA with various concentrations of quercetin.

553

Figure 3. Infrared spectra of native and glycated BSA with various concentrations of quercetin.

554

Figure 4. SDS-PAGE patterns (A) and MALDI-TOF mass spectra (B) of native BSA and

555


556

Figure 5. The mass spectra of representative glycated peptides of BSA after trypsin digestion. (A)

557

peptide 184-204 at m/z 1244.05802+; (B) peptide 569-587 at m/z 1103.49533+; (C) peptide

558

264-285 at m/z 653.79454+; (D) peptide 524-544 at m/z 833.40153+. The mass shifts between the

559

glycated and unglycated peptides are indicated above the arrows.

560

Figure 6. HCD MS/MS spectra of the representative glycated peptides of BSA after trypsin

561

digestion. (A) glycated peptide 89-105 with m/z of 527.77314+; (B) glycated peptide 401-420 with

562

m/z of 839.73573+; (C) glycated peptide 372-386 with m/z of 988.93882+. The sequence of each

563

peptide is shown on the right top of each spectrum. The determined glycation sites are indicated

564

by a line with fructose. The b and y ions are indicated in the spectra. CAM refer to

565

carbamidomethyl.

566

Figure 7. The DPPH· scavenging ability (A), ABTS·+ scavenging ability (B), emulsifying activity

567

index (C) and emulsion stability (D) of native and glycated BSA with and without quercetin.

568



Page 28 of 36

569

Table 1

570

The glycated peptides and glycation sites of glycated BSA with various concentrations of quercetin. Native

Peptide

Glycated

Mass

peptide

location

peptide

shift

1

398.5383+

25-34

452.55473+

2

417.21143+

35-44

471.2293+

3

487.26044+

89-105

527.77304+

4

627.46125+

5

847.72513+

6

NO.

106-130

659.87075+

Glycated

BSA

site

-Fru

54.0167 (R)DTHKSEIAHR(F)

K28

+

-

+

+

54.0176 (R)FKDLGEEHFK(G)

K36

+

+

+

+

40.5125 (K)SLHTLFGDELC*KVASLR(E)

K100

+

+

+

+ -

sequence

Que0.5

Que1.5 Que3.0

32.4095 (R)ETYGDMADC*C*EKQEPERNEC*FLSHK(D)

K117

+

-

-

118-138

901.74223+

54.0171 (K)QEPERNEC*FLSHKDDSPDLPK(L)

K130

+

+

+

-

788.88672+ 139-151

869.91282+

81.0261 (K)LKPDPNTLC*DEFK(A)

K140

+

+

+

+

7

673.99353+ 139-155

728.01093+

54.0174 (K)LKPDPNTLC*DEFKADEK(K)

K140

+

+

+

+

8

476.22264+ 173-188

516.73584+

40.5132 (R)NEC*FLSHKDDSPDLPK(L)

K180

+

+

-

+

9

1244.05822+ 184-204 1325.08492+

81.0267 (K)YNGVFQEC*C*QAEDKGAC*LLPK(I)

K197

+

+

+

-

10

424.25512+

242-248

505.28172+

81.0266 (R)LSQKFPK(A)

K245

-

-

-

+

11

647.85442+ 246-256

728.88092+

81.0265 (K)FPKAEFVEVTK(L)

K248

-

+

+

-

12

1138.47002+ 264-280 1057.44462+

81.0270 (K)VHKEC*C*HGDLLEC*ADDR(A)

K266

+

+

+

+

13

647.98103+

281-297

701.99553+

54.0145 (R)ADLAKYIC*DNQDTISSK(L)

K285

-

-

+

-

14

766.89292+

298-309

847.91922+

81.0263 (K)LKEC*C*DKPLLEK(S)

K299

+

+

-

+

15

646.30382+

300-309

727.33092+

81.0271 (K)EC*C*DKPLLEK(S)

K304

+

+

-

-

16

1171.22823+

309-340

1225.24653+

54.0183 (K)SHC*IAEVEKDAIPENLPPLTADFAEDKDVC*K(N)

K317

+

+

+

+

17

1151.04522+

341-359

1232.07052+

81.0253 (K)NYQEAKDAFLGSFLYEYSR(R)

K346

-

+

+

+

18

1013.42063+

375-399

1067.44003+

54.0187 (K)EYEATLEEC*C*AKDDPHAC*YSTVFDK(L)

K386

+

-

-

-

19

898.41802+ 387-401

979.44452+

81.0265 (K)DDPHAC*YSTVFDKLK(H)

K399

-

-

-

+

20

773.95012+ 400-412

854.97642+

81.0263 (K)LKHLVDEPQNLIK(Q)

K401

-

+

-

+

21

785.71813+ 401-420

839.73573+

54.0176 (K)HLVDEPQNLIKQNC*DQFEK(L)

K411

+

+

-

-

22

389.50223+ 460-468

443.52003+

54.0178 (R)C*C*TKPESER(M)

K463

+

+

+

-

23

770.41342+

483-495

851.43872+

81.0253 (R)LC*VLHEKTPVSEK(V)

K489

-

+

+

-

24

733.85632+

496-507

814.88382+

81.0275 (K)VTKC*C*TESLVNR(R)

K498

+

+

+

+

25

824.39903+

508-528

878.41843+

54.0194 (R)RPC*FSALTPDETYVPKAFDEK(L)

K523

-

+

+

+

26

833.40153+

524-544

887.41863+

54.0171 (K)AFDEKLFTFHADIC*TLPDTEK(Q)

K528

+

+

+

+

27

759.72383+

529-547

813.74123+

54.0174 (E)LFTFHADIC*TLPDTEKQIK(K)

K542

-

+

-

+

28

436.91433+

558-568

490.93193+

54.0176 (K)HKPKATEEQLK(T)

K559/561

+

-

+

-

29

733.70503+ 562-580

787.72203+

54.0170 (K)ATEEQLKTVMENFVAFVDK(C*)

K568

-

+

-

+

30

1103.49533+ 569-597 1157.51273+

54.0174 (K)TVMENFVAFVDKC*C*AADDKEAC*FAVEGPK(L)

K580

+

+

+

+

31

964.40122+ 581-597 1045.42822+

81.0270 (K)C*C*AADDKEAC*FAVEGPK(L)

K587

+

+

+

+

571

C* means the cystine residue alkylated by carbamidomethyl. BSA-Fru: BSA glycated without quercetin; Que0.5:

572

BSA glycated with 0.5 mM quercetin; Que1.5: BSA glycated with 1.5 mM quercetin; Que3.0: BSA glycated with

573

3.0 mM quercetin.


Page 29 of 36


Fluorescence intensity

250

200

150

100

50

575

Que 0

Que 0.5 Que 1.0 Que 1.5 Que 2.0 Que 3.0

AG

Samples

576

Figure 1. Effect of quercetin on the fluorescence intensity of glycated BSA at an excitation and

577

emission wavelength of 350 nm and 425 nm, respectively.

578



A 10000

Glycated BSA Native BSA Que 0.5 Que 1.5 Que 3.0 Que 0.5B Que 1.5B Que 3.0B

8000

6000

4000

B

10000



8000

Page 30 of 36

Glycated Native

6000

4000

2000 2000

0

300

579

320

340

360

Samples

380

400

BSA

Que 0.5

Que 1.5

Samples

Que 3.0

580

Figure 2. Intrinsic fluorescence spectra (A) and maximum fluorescence intensity (B) of native and

581


582


Page 31 of 36


Que3.0B Que1.5B

Que0.5B Que3.0 Que1.5

Que0.5 586.3-621

1060.7

2933.2

2962.1

BSA-Fru 1085.7

BSA 1546.6

400

800

3388.3

1200

3430.7

1654.6

1600

2000

2400

2800

3200

3600

4000

583

Wavenumbers (cm-1)

584

Figure 3. Infrared spectra of native and glycated BSA with various concentrations of quercetin.



A

Page 32 of 36

B 66876.6328 33478.9336

33619.2578 33633.2813

34423.3125 33368.1953

20000

40000

80813.8516

Que 3.0

67097.4141 81094.8528

Que 1.5

81133.7969

Que 0.5

81506.8828

BSA-Fru

80446.8750

BSA

67140.7266 67496.8828

66613.8828

60000

80000

Mass (m/z)

586 587

Figure 4. SDS-PAGE patterns (A) and MALDI-TOF mass spectra (B) of native and glycated BSA with and

588

without quercetin.

589


100000

Page 33 of 36


Relative intensity

80 60

mass shift= 81.0270

40 1325.0850 z=2

20 1240 C

Relative intensity

100

1260

m/z

1300

1320

1103.4953 z=3

60 40

mass shift= 54.0174

20

1340

1100

1120

m/z

1140

40 mass shift= 40.51414

660

670

680

m/z

694.3086 z=4

690

700

80 60

mass shift= 54.0171

40 833.4015 z=3

20

710

1160 887.4186 z=3

100

60

650

80

D

80

640

590

1280

653.7945 z=4

20

1157.5127 z=3

B 100

1244.0580 z=2

Relative intensity

Relative intensity

A 100

825

840

855

m/z

870

885

900

591

Figure 5. The mass spectra of representative glycated peptides of BSA after trypsin digestion. (A) peptide 184-204

592

at m/z 1244.05802+; (B) peptide 569-587 at m/z 1103.49533+; (C) peptide 264-285 at m/z 653.79454+; (D) peptide

593

524-544 at m/z 833.40153+. The mass shifts between the glycated and unglycated peptides are indicated above the

594

arrows.

595


Abundance 4 ×10 16.0

12.0

8.0

4.0

200 400

598 600

m/z 800

m/z 800 1000

12 11 10 9 8 7 6 5 4

1000

ACS Paragon Plus Environment 1200

1340.5790, y9+-H2O 1453.6431, y10+-H2O

1000

1142.5726 b10+-NH3 1159.6230, b10+

800 1200

1

1200

L A K E Y E A T L E E C(CAM) C(CAM) A K 3

1539.7410, y12+-NH3 1556.7460, y12+

1391.6768, y11+-H2O 1409.6927, y11+

1237.6476, y9+ 1273.5880, b11+ 1334.6544, y10+-H2O 1352.6810, y10+

1108.6030, y8+ 1113.5582, b10+

977.5001, y7+-H2O 995.5179, y7+ 1000.4719, b9+

778.8842, y122+ 817.4299, y6+-H2O 835.4991, y6+ 871.4299, b8+

699.3824, b6+ 705.3491, y112+

545,3400, y5+

6 5 4 3

1373.5746, y11+

b

8 9 10

1192.4879, y10+-H O 2 1210.5088, y10+

m/z

1010.4259, y8+

600

2 3 4 5 6

1081.4661, y9+

b

909.3801, y7+

600

801.3859, b7+-H2O 819.3997, b7+ 826.3391, y6+ 916.4248, b8+-NH3 923.3487, y7+-NH3 933.4424, b8+ 940.3806, y7+ 1051.4153, y8+-NH3 1046.5233, b9+ 1068.4410, y8+

596

796.2925, y6+

400 666.3138, y5 691.3386, b6+

594.2877, b5+

3+ B ×105 [M+H] =839.7357

+

552,3130, b5+

446.2717, y4+

439.2294, b4+

338.1817, b3+ 375.2346, y3+

320.1717, b3+-H2O

12 11 10 9 8 7

S L H T L F G D E L C(CAM) K V A S L R

10 9 8 7 6 5 4 3 2 1

2

1502.6100, y12+

[M+H]2+=988.9388 534.2557, y4+-NH3 551.2813, y4+

465.2450, b4+

423.2245, y3+

350.2182, b3+

400

b

788.2875, y6+-H2O

C 251.1503, b2+

0.5

649.2449, y5+-H2O 667.2516, y5+

200

538.2105, y4+

597 175.1189, y1+

4+ A 6 [M+H] =527.7731 ×10

455.2504, y72+

3.0

378.1802, y3

6.0

276.1559, y2+

200

+

9.0 201.1232, b2+

1.0

325.1859, y52+-H O 2

12.0

147.1126, y1+-NH3 130.0863, y1+

Abundance 1.5

147.1126, y1+ 185.1278, b2+ 218.1498, y2+

Abundance

Journal of Agricultural and Food Chemistry Page 34 of 36

y

fructose

1400 1600

HLVDEPQNLIKQNCDQFEK

y

2 3 4 5 6 7 8 9 10 fructose

1400 y

2

fructose

1400

599 Figure 6. HCD MS/MS spectra of the representative glycated peptides of BSA after trypsin digestion. (A)

600 glycated peptide 89-105 with m/z of 527.77314+; (B) glycated peptide 401-420 with m/z of 839.73573+; (C)

601

glycated peptide 372-386 with m/z of 988.93882+. The sequence of each peptide is shown on the right top of each

602

spectrum. The determined glycation sites are indicated by a line with fructose. The b and y ions are indicated in the

603

spectra. CAM refers to carbamidomethyl.


80 70 60 50 40 30 20 10

C

700

Emulsifying activity index (m2/g)

0

600

Que0

Que0.5

Que1.5

B

Samples Glycated BSA Native BSA

60 50 40 30 20 10

Emulsion stability (%)

300 200 100 Que0.5

Que1.5

Que3.0

Que0

Que0.5

Que1.5

Que3.0

Samples

70 60

400

Que0

70

0

D

Glycated BSA Native BSA

80

Que3.0

500

0

605


ABTS·+ scavenging ability (%)

A DPPH· scavenging ability (%)

Page 35 of 36


50 40 30 20 10 0

Que0

Que0.5

Que1.5

Que3.0

Samples

Samples

606

Figure 7. The DPPH· scavenging ability (A), ABTS·+ scavenging ability (B), emulsifying activity index (C) and

607

emulsion stability (D) of native and glycated BSA with and without quercetin.



TOC graphic

608

Glycation

＋

Intrinsic fluorescence

OH

O CH2 OH OH OH OH

609

Molecular weight

OH

OH

HO

O OH OH O

Quercetin

Glycated peptides and glycation sites


Page 36 of 36

Insights into the Mechanism of Quercetin against BSA-Fructose

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