Mechanism of Reduction in IgG and IgE Binding of ... - ACS Publications

Aug 11, 2017 - In this work, the effect of ultrasound pretreatment combined with dry-state glycation on the IgG and IgE binding of β-Lg was studied. ...
0 downloads 0 Views 2MB Size
Subscriber access provided by Eastern Michigan University | Bruce T. Halle Library

Article

Mechanism of the reduction in the IgG and IgE binding of #-lactoglobulin induced by ultrasound pretreatment combined with dry-state glycation: a study using conventional spectrometry and high resolution mass spectrometry Wenhua Yang, Zongcai Tu, Hui Wang, Lu Zhang, Shengsheng Xu, Chendi Niu, Honglin Yao, and Igor A. Kaltashov J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02842 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 18, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

Journal of Agricultural and Food Chemistry

1

Mechanism of the reduction in the IgG and IgE binding of β-lactoglobulin

2

induced by ultrasound pretreatment combined with dry-state glycation: a study

3

using conventional spectrometry and high resolution mass spectrometry

4

Wenhua Yang, Zongcai Tu,

5

Honglin Yao, Igor A. Kaltashov

6 7 8 9 10 11



#



*, †, §

*, †

Hui Wang,

§

#

Lu Zhang, Shengsheng Xu, Chendi Niu,

#

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

330047, China §

#

*

#

College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi, 330022, China Department of Chemistry, University of Massachusetts-Amherst, Amherst, MA, 01003, USA Corresponding authors. Tel: +86 -791-8812-1868; Fax: +86-791-8830-5938.

E-mail: [email protected] (Zongcai Tu); [email protected] (Hui Wang).

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

12

Abstract: Bovine β-lactoglobulin (β-Lg) is one of major allergens in cow's milk. Previous study

13

showed that ultrasound treatment induced the conformational changes of β-Lg and promoted the

14

glycation in aqueous solutions, which is, however, less efficient compared with dry-state. In this

15

work, the effect of ultrasound pretreatment combined with dry-state glycation on the IgG and IgE

16

binding of β-Lg was studied. Dry-state glycation with mannose after ultrasound pretreatment at

17

0-600 W significantly reduced the IgG and IgE binding of β-Lg, with the lowest values observed

18

at 400 W. The decrease in the IgG and IgE binding of β-Lg was attributed to the increase in

19

glycation extent and the changes of secondary and tertiary structure, which reflected in the

20

increase of UV absorbance, α-helix and β-sheet contents as well as the decrease of intrinsic

21

fluorescence intensity, surface hydrophobicity, β-turn and random coil contents. Moreover,

22

ultrasound pretreatment promoted the reduction of IgG and IgE binding abilities by improving

23

glycation, reflecting in the increase of the glycation sites and the degree of substitution per peptide

24

(DSP) value determined by Fourier transform ion cyclotron resonance mass spectrometry

25

(FTICR-MS). Ultrasound pretreatment at 400 W showed the most significantly enhanced

26

glycation extent. Besides, the results suggested FTICR-MS could provide insights into the

27

glycation at molecular level, which was conducive to the understanding of the mechanism of the

28

reduction in the IgG and IgE binding of β-Lg. Therefore, ultrasound pretreatment combined with

29

dry-state glycation may be a promising method for β-Lg hyposensitization.

30

Keywords: β-lactoglobulin; IgG and IgE binding abilities; ultrasound pretreatment; dry-state

31

glycation; FTICR-MS

2

ACS Paragon Plus Environment

Page 2 of 40

Page 3 of 40

Journal of Agricultural and Food Chemistry

32

ABBREVIATIONS USED

33

β-Lg, β-lactoglobulin; IgE, immunoglobulin E; IgG, immunoglobulin G; ELISA, enzyme-linked

34

immunosorbent assay; OVA, ovalbumin; BSA, bovine serum albumin; CD, circular dichroism;

35

UV, ultraviolet; DTT, dithiothreitol; ANS, 1-anilinonaphthalene-8-sulfonate; PBS, phosphate

36

buffer solution; PBST, PBS with 0.05% Tween; DSP, degree of substitute per peptide; HPLC,

37

high performance liquid chromatography; FTICR-MS, Fourier transform ion cyclotron resonance

38

mass spectroscopy; ECD, electron capture dissociation.

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

39

Page 4 of 40

Introduction

40

Bovine β-lactoglobulin (β-Lg), constitutes about 60% of whey proteins, is the major allergen

41

that causes about 90% IgE-mediated cow's milk allergy (CMA).1, 2 It is a 162-amino-acid globular

42

protein with a molecular mass of 18.36 kDa.3 The formation of two disulfide bonds

43

(Cys66-Cys160 and Cys106-Cys119) stabilizes a so-called-barrel (or calyx) structure that formed

44

by nine anti-parallel-sheets.4 So far, its IgG and IgE epitopes, including linear and conformational

45

types, are mainly identified.5, 6 Recently, numerous methods have been investigated to alter the

46

allergenicity of β-Lg through physical modifications, such as heating,7 dynamic high pressure

47

microfluidization (DHPM),8 high hydrostatic pressure9 and high-intensity ultrasound treatment;10

48

chemical treatments, such as hydrolysis11,

49

However, DHPM and high hydrostatic pressure could increase the allergenicity of β-Lg, whereas

50

high-intensity ultrasound treatment had insignificant effect on the allergenicity of β-Lg.

51

Hydrolysis has some sensory defects (such as bitterness) and might cause some potential

52

disordered metabolism.11 Genetic modification is controversial and inefficient.16

12

and glycation;13 and genetic modification.14,

15

53

Among them, glycation has been considered as a safe way for modifying proteins compared

54

with other methods.17 It generally takes place between proteins and reducing sugars during the

55

first stage of the Maillard reaction. As one of most common and important chemical reactions

56

during food processing and storage, glycation is often used to modify the food protein property,

57

such as antioxidant activity and allergenicity.18,

58

decrease the allergenicity to a satisfactory result due to the insufficient glycation extent.

59

19

However, a single modification could not

High-intensity ultrasound is an emerging non-thermal technology that uses a collective force of 4

ACS Paragon Plus Environment

Page 5 of 40

Journal of Agricultural and Food Chemistry

60

high temperature, high pressure, microjet, shear force, shock wave and turbulence.20 Our previous

61

work showed that ultrasound treatment partially unfolded the structure of ovalbumin (OVA) and

62

promoted the glycation of bovine serum albumin (BSA).21, 22 Stanic-Vucinic et al.18 reported that

63

ultrasound promoted the glycation of β-Lg in aqueous solutions. Therefore, ultrasound

64

pretreatment combined with glycation may provide a new method for β-Lg desensitization.

65

However, the glycation efficiency at dry-state is far higher than that in aqueous solutions.22 Few

66

studies investigated the influence of ultrasound pretreatment coupled with dry-state glycation on

67

allergenicity of β-Lg. Moreover, the mechanism of the decrease in allergenicity of β-Lg by

68

glycation after ultrasound pretreatment remains ambiguous.

69

The purpose of this study was therefore to carry out a detailed interpretation of the relationship

70

between allergenicity and structural changes of β-Lg induced by ultrasonic pretreatment combined

71

with dry-state glycation. In this work, β-Lg was pretreated by high-intensity ultrasound followed

72

by glycated with mannose at dry-state under non-denatured condition. The IgG and IgE binding of

73

β-Lg samples were evaluated by inhibition ELISA. The structural changes were characterized by

74

the conventional spectrometry, including circular dichroism (CD), ultraviolet (UV) and

75

fluorescence spectrometry. The glycation sites and extent of glycated β-Lg were determined by

76

Fourier transform ion cyclotron resonance mass spectroscopy (FTICR-MS). The results of this

77

research enhance our understanding of the mechanism of the decrease in IgG and IgE binding of

78

β-Lg induced by ultrasound pretreatment combined with dry-state glycation, as well as

79

demonstrating that ultrasound pretreatment combined with dry-state glycation may be a promising

80

method for β-Lg hyposensitization.

5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

81

Materials and methods

82

Materials

83

Page 6 of 40

β-Lg (L3908), D-(+)-mannose (M2069), goat anti-rabbit IgG-HRP conjugate (A9169) and goat

84

anti-human

IgE-HRP conjugate

(A9667),

1-Anilinonaphthalene-8-sulfonate (ANS)

and

85

3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

86

CMA patients' sera were bought from PlasmaLab International (Everett, W.A., USA) and frozen

87

(-80 °C) until analysis. Their specific IgE levels ranged from 9.58 kU L-1 to 96.6 kU L-1 (detailed

88

information is shown in Figure S1). The polyclonal antisera against β-Lg were obtained from 6

89

young (three months old, about 2.0 kg) Japanese male rabbits. H2O used in this work were Milli-Q

90

water. All chemicals used were of analytical grade.

91

β-Lg samples preparation

92

β-Lg solution (1 mg/mL, dissolved in 50 mmol L-1 phosphate buffered saline (PBS), pH 8.0)

93

was placed in a beaker to be treated by a sonicator (JY98-ⅢDN, Scientz Biotechnology Co. Ltd.,

94

Ningbo, China). The frequency is 20 kHz and the maximum power is 950 W. The solution was

95

irradiated with an ultrasonic wave directly from the probe with a vibrating titanium tip of 15 mm.

96

Samples were treated for 30 min at different powers (0, 200, 400 and 600 W). The ultrasonic

97

intensity at 200, 400 and 600 W was 38 - 42 W cm-1, 57 - 59 W cm-1 and 68 - 72 W cm-1,

98

respectively. The sonicator worked for 3 s after the pause of 7 s. The samples were treated in the

99

ice bath to keep the processing temperature below 20 °C. Then the mannose was added into the

100

samples (the mass ratio of β-Lg and mannose was 1:2). After mixing evenly, all the samples (100 6

ACS Paragon Plus Environment

Page 7 of 40

Journal of Agricultural and Food Chemistry

101

µL, 10 µg mL−1) were lyophilized and incubated for 4 h at 55 °C and 0.79 water activity. Then

102

they were placed into the freezer to stop the reaction. The remaining mannose was removed by

103

Millipore ultrafiltrator (molecular weight cut off 3,000 Da). Finally, all samples were adjusted to 1

104

µg mL−1 and stored at 4 °C for less than 48 h. Native β-Lg was a control and named β-Lg-N. The

105

sample without mannose heated under the same glycation conditions was another control and

106

named β-Lg-H. The glycated samples with ultrasound pretreatment at 0, 200, 400 and 600 W were

107

named β-Lg-M, β-Lg-M-200, β-Lg-M-400 and β-Lg-M-600, respectively.

108

Determination of IgG and IgE binding abilities

109

The IgG and IgE binding of β-Lg samples was evaluated by inhibition ELISA with rabbit

110

antisera and CAM patients' antisera, respectively.23 Firstly, the 96-well microplate was coated

111

overnight at 4 °C with native β-Lg (100 µL per well, 2 µg mL−1). After washing with PBST

112

(0.05% Tween-20 in 50 mmol L-1 PBS, pH 7.4), the microplate was blocked with 50 mg mL-1

113

skimmed milk for 1 h at 37 °C. Then 50 µL of either pooled rabbit antisera (diluted to1:12,800) or

114

pooled CAM patients' antisera (diluted to 1:8) and 50 µL β-Lg samples (inhibitors) were added

115

after washing. After incubation for 1 h at 37 °C, it was washed for 5 times with PBST. Then 100

116

µL of goat anti-human IgE-HRP conjugate or goat anti-rabbit IgG-HRP conjugate (diluted to

117

1:5,000 in PBST) was added and incubated for 30 min at 37 °C. After washing, 100 µL of TMB

118

solution was added and incubated for 15 min at 37 °C. Finally, 50 µL of 2 mol L-1 sulfuric acid

119

was added to stop the reaction and the absorbance was measured at 450 nm using a microplate

120

reader (HF2000, Huaan Magnech, Beijing, China). The inhibition rate was calculated using the

121

following equation: Inhibition = (1 − B / B0) × 100%, where B and B0 are the absorbance values 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

122

of the well with and without the inhibitor, respectively. IC50 is the concentration of inhibitors that

123

causes a 50% inhibition of antibody binding (µg mL-1).

124

Far-UV CD spectroscopy

125

Far-UV (190-240 nm) CD spectroscopy was performed to define the secondary structure of

126

β-Lg samples. The CD spectra of β-Lg (0.1 mg mL-1 in 50 mmol L-1 PBS, pH 8.0) were collected

127

by using a MOS-450 spectropolarimeter (Bio-Logic SAS, Claix, French). The path length,

128

bandwidth and step resolution were 1.0 mm. The scan speed was 100 nm min-1. The data were

129

shown in terms of molar residue ellipticity ([θ], deg cm2 dmol-1), and the contents of different

130

secondary structures were analyzed with DichroWeb.

131

UV absorption and intrinsic fluorescence spectroscopy

132

UV absorption spectrum was performed by Hitachi spectrophotometer (UV-2910, Hitachi,

133

Tokyo, Japan). The β-Lg samples (0.5 mg mL-1 in 50 mmol L-1 PBS, pH 8.0) were scanned from

134

240 to 320 nm at a scan speed of 800 nm min-1. Intrinsic fluorescence was measured by Hitachi

135

spectrofluorimeter (F-7000, Hitachi, Tokyo, Japan). The concentration of β-Lg was 0.1 mg mL-1.

136

The emission spectra were scanned from 300 nm to 420 nm at a speed of 1200 nm min-1 with the

137

excitation wavelength of 280 nm. The bandwidth, excitation and emission slits were all 5.0 nm.

138

Surface hydrophobicity

139 140

According to the method of Matulis and Lovrien

24

with modification, the surface

hydrophobicity of β-Lg samples was measured using ANS fluorescent probes. The volume ratio of 8

ACS Paragon Plus Environment

Page 8 of 40

Page 9 of 40

Journal of Agricultural and Food Chemistry

141

sample (0.25 mg mL-1, 0.5 mg mL-1 and 1.0 mg mL-1) and ANS solution (0.008 mol L-1) was

142

200:1. The emission spectrum was scanned from 400 to 600 nm at a speed of 1200 nm min-1 with

143

the excitation wavelength of 390 nm. Other parameters were same as that of intrinsic fluorescence

144

measurement. The surface hydrophobicity of the samples was defined as the ratio of the extrinsic

145

fluorescence intensity to the protein concentration with different concentrations.

146

HPLC-FTICR-MS analysis

147

Firstly, β-Lg (10 µg) was added into the Tris-HCl buffer (pH 7.5, 1 mol L-1) containing 6 mol

148

L-1 guanidine. Then 0.25 µL of 0.5 mol L-1 DTT was added and incubated for 30 min at 37 °C,

149

following by adding of 0.5 µL iodoacetamide (0.5 mol L-1). After dark incubation for 30 min at

150

room temperature, the solution was diluted 10-fold with H2O. Then 2 µL of 1 mg mL-1 trypsin was

151

added and incubation overnight at 37 °C. Next, 2 µL of 10% formic acid (FA) was added to stop

152

the reaction. Then 50 µL of sample solution was injected into a BioSuite C18 column (2.1 mm ×

153

50 mm, 3µm; Waters, Milford, MA) for HPLC-MS. The flow rate of LC was 0.2 mL min-1. The

154

mobile phase consisted of solvent A (0.1% FA in H2O) and solvent B (100% acetonitrile

155

containing 0.1% FA). The gradient elution followed as: 0-5 min, 5% solvent B; 5-35 min, 5%-50%

156

solvent B gradient; 35-37 min, 60%-80% solvent B gradient; 37-40 min, 80% solvent B; 40-42,

157

20%-95% solvent A gradient. The effluent was infused into the Bruker 7T-SolariX FTICR-MS

158

(Bruker Daltonics Inc., Billerica, MA). The electron capture dissociation (ECD) fragmentation

159

mode was used to acquire MS/MS spectra. The ionization energy was 1.0 eV and the capillary

160

voltage was 4500 V. The temperature and flow rate of dry gas was 180 °C and 8 L min-1,

161

respectively. The software (DataAnalysis and BioTools) were used to identify peptides according 9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

162 163 164

to the combination of accurate masses and MS/MS spectra. To further compare the glycation extent of each peptide, the average degree of substitution per peptide (DSP) was calculated according to the following formulation: 25 n

165

DSP =

∑ i×I ∑ I i −0 n

( peptide +i ×mannose )

i = 0 ( peptide+ i ×mannose )

166

where I is the sum of the intensities of the glycated peptides and i is the number of mannose units

167

attached to the peptide in each glycated form.

168

Statistical analysis

169

All the experiments were carried out in triplicate and the results were presented as mean value ±

170

standard deviation (SD). The analysis was performed using Origin-Pro 2017 (OriginLab Corp.,

171

Northampton, MA).

172

Results and discussion

173

IgG and IgE binding capacities analysis

174

The IgG and IgE binding abilities of β-Lg samples were determined by inhibition ELISA with

175

anti-β-Lg-sera from rabbit and CMA patients, respectively. The ability of antibody binding to

176

allergen was reflected by the IC50 value. Higher IC50 value implies lower binding capacity. Figure

177

1 shows the effect of ultrasound pretreatment combined with glycation on the IgG and IgE binding

178

of β-Lg. As shown in Figure 1A, compared with β-Lg-N (1.94 µg mL-1), β-Lg-H showed almost

179

the same IC50 value, whereas the glycated β-Lg samples exhibited much higher IC50 values, with

180

highest value (18.11 µg mL-1, about 9-fold with β-Lg-N) observed at 400 W. When β-Lg was 10

ACS Paragon Plus Environment

Page 10 of 40

Page 11 of 40

Journal of Agricultural and Food Chemistry

181

glycated after ultrasound pretreatment at 0-400 W, the IC50 value of IgG gradually increased with

182

increasing ultrasound intensity. However, when it was glycated after ultrasound pretreatment at

183

600 W, the IC50 value decreased to 13.75 µg mL-1. Similar results were observed in Figure 4B. At

184

ultrasound power of 0 (7.89 µg mL-1), 200 (11.86 µg mL-1), 400 (20.07 µg mL-1), and 600 (14.82

185

µg mL-1)W, the IC50 values of glycated β-Lg were 2.95-, 5.00-, 7.74-, and 5.55-fold higher than

186

that of β-Lg-H (2.67 µg mL-1). These results indicated that glycation significantly reduced the IgG

187

and IgE binding of β-Lg and ultrasound pretreatment promoted the reduction. It was also similar

188

to that reported in a previous study,13, 23, 26 wherein they showed that glycation notably decreased

189

the IgG and/or IgE binding of β-Lg.

190

The decrease in IgG and IgE binding abilities may be attributed to the shielding of some

191

epitopes by the covalent attachment of amino acid residues and reducing sugar. Moreover, the

192

changes in conformational structure induced by glycation may be responsible for it. Although

193

high-intensity ultrasound treatment hardly affected the allergenicity of β-Lg, it caused the

194

significant changes in conformational structure of β-Lg.10 Our previous work showed that the

195

conformational structure of OVA was partially unfolded by ultrasound treatment.21 Moreover, the

196

glycation of BSA was improved and the glycation sites were dramatically increased by ultrasound

197

pretreatment.22 These may be the reasons that ultrasound pretreatment resulted in the further

198

reduction of the IgG and IgE binding of glycated β-Lg. However, too high-intensity ultrasound

199

pretreatment could make the proteins refold and some epitopes buried, leading the decrease in the

200

IgG and IgE binding abilities.21 Therefore, the decrease in the IgG and IgE binding of β-Lg in this

201

work may result from both the masking of some epitopes and the conformational changes induced

202

by ultrasound pretreatment combined with glycation. In order to better understand the mechanism 11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

203

of the reduction in IgG and IgE binding of β-Lg by ultrasound pretreatment combined with

204

glycation, the conformation changes of β-Lg were monitored by conventional spectroscopy and

205

the glycation extent and sites were determined by FTICR-MS in the subsequent experiments.

206

Secondary structure analysis

207

The changes in CD spectra of β-Lg induced by ultrasound pretreatment combined with

208

glycation are shown in Figure 2 and Table 1. There were no significant changes in the secondary

209

structure of β-Lg after heating without mannose at dry-state (p < 0.05), suggesting that dry-state

210

heating under non-denatured conditions (55 °C for 4 h) had little influence on the secondary

211

structure of β-Lg. It may result from the limited movement of protein molecule due to the low

212

water activity. Similar results were found by Matsudomi et al.,

213

dry-heated at 80 °C for 7 days had nearly the same secondary structure with untreated OVA. After

214

glycation, the α-helix and β-sheet contents of β-Lg increased from 52.9% (β-Lg) to 58.4%

215

(β-Lg-M-400) while the β-turn and random coil contents decreased from 47.1% (β-Lg) to 41.6%

216

(β-Lg-M). It suggested that glycated β-Lg was partially unfolded and reorganized to be a more

217

stable structure. Similar results were also reported that glycation increased the α-helix and β-sheet

218

contents and decreased β-turn and random coil contents.13 Glycation could make conformation of

219

protein to a more stable state at the expense of β-turn or unordered structure.28 It may result in the

220

increase of α-helix and β-sheet contents and the decrease of β-turn and random coil contents due

221

to the glycation. However, the result was opposite to other studies,18, 29 in which they showed no

222

significant changes induced by glycation. It may be caused by the different glycation conditions,

223

such as the sugar used, water activity, pH, reaction temperature and time. Moreover, ultrasound 12

ACS Paragon Plus Environment

27

who showed that OVA

Page 12 of 40

Page 13 of 40

Journal of Agricultural and Food Chemistry

224

pretreatment induced further increase of α-helix and β-sheet contents and decrease of β-turn and

225

random coil contents. It may be attributed to the promotion of glycation by ultrasound

226

pretreatment. The results implied the secondary structure of β-Lg was significantly changed by

227

ultrasound pretreatment combined with dry-state glycation.

228

UV absorption and intrinsic fluorescence spectra analysis

229

The changes in UV absorption spectra of β-Lg samples induced by ultrasound pretreatment

230

combined with glycation are shown in Figure 3A. Compared to β-Lg-N, β-Lg-H had a very slight

231

increase in the maximum UV absorbance at 280 nm, where as β-Lg-M had a notable increase from

232

0.47 (β-Lg) to 0.55 (β-Lg-M). Moreover, the glycated samples with ultrasound pretreatment had

233

higher maximum UV absorbance at 280 nm and β-Lg-M-400 had the highest value. It implied that

234

glycation could partially unfold structure of β-Lg and make aromatic amino acid (mainly Trp)

235

residues exposed on the surface of β-Lg molecule. The results may be attributed to the destruction

236

of the interactions that maintain the rigid native protein tertiary structure by ultrasound

237

pretreatment coupled with dry-state glycation. Previous study also showed similar results that

238

ultrasound-induced glycation of β-Lg made the tertiary structure of β-Lg surrounding Trp19

239

markedly loosened.18

240

The intrinsic tryptophan fluorescence spectra of native and glycated β-Lg are shown in Figure

241

3B. Similarly, compared with β-Lg-N, β-Lg-H had a very slight increase in the intensity of

242

fluorescence emission maximum (λmax). However, when β-Lg was glycated, the λmax intensity

243

decreased from 842 (β-Lg-N) to 513 (β-Lg-M), and the glycated β-Lg after ultrasound

244

pretreatment showed lower λmax intensity, with a lowest value λmax intensity of 386 13

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

245

(β-Lg-M-400), indicating that conformational structure of β-Lg was dramatically changed by

246

ultrasound pretreatment combined with glycation. It may result from the quenching of

247

fluorescence intensity induced by the exposure of Trp residues to solvent. Moreover, a red shift of

248

λmax from 340 nm (native β-Lg) to 350 nm (glycated β-Lg) implied the polarity of β-Lg was

249

changes. The result was also similar to some reports,23, 30 wherein they showed that glycation

250

significantly decreased the intrinsic fluorescence intensity and induced a red shift of λmax in

251

fluorescence spectra of β-Lg.

252

Surface hydrophobicity

253

The surface hydrophobicity of β-Lg samples are shown in Figure 3C. β-Lg-H had no

254

significant difference in the surface hydrophobicity with β-Lg-N (p < 0.05). When β-Lg was

255

glycated with mannose at ultrasound pretreatment at 0-600 W, the surface hydrophobicity declined

256

from 1673 (β-Lg-N) to 1465 (β-Lg-M), 1352 (β-Lg-M-200) 1280 (β-Lg-M-400) and

257

1333(β-Lg-M-600), respectively. The result was in accordance with previous reports that glycation

258

significantly reduced the hydrophobicity of β-Lg.23, 30 It may be attributed to the masking of some

259

hydrophobic groups induced by amino acid residues modification through covalent attachment of

260

mannose. What's more, ANS could strongly bind cationic groups, such as lysine (Lys) and

261

arginine (Arg) residues.31 The interaction of Lys and/or Arg residues and mannose by glycation

262

also led to the decrease of surface hydrophobicity of β-Lg. Ultrasound pretreatment could promote

263

conjugation of Lys and/or Arg residues with mannose, make ANS bind less cationic groups and

264

finally lead to the further reduction of surface hydrophobicity. Accordingly, glycated β-Lg with

265

ultrasound pretreatment had lower surface hydrophobicity than that without ultrasound 14

ACS Paragon Plus Environment

Page 14 of 40

Page 15 of 40

Journal of Agricultural and Food Chemistry

266

pretreatment.

267

Glycation site determination

268

To acquire MS/MS spectra, ECD fragmentation mode produced sufficient c and z ions was used

269

in this work due to the retention of glycation sites during MS. Trypsin was chosen as the digestion

270

enzyme due to the specifically cleavage on the carbon side of amino acids Lys and Arg. It cannot

271

cleave the site when the Lys or Arg is modified by glycation. To reduce the formation of

272

aggregation, the glycation was performed at 55 °C for 4 h (pH 8.0) under dry-state. No apparent

273

protein aggregation happened under this condition. The result above showed that dry-heating

274

without mannose at 55 °C for 4 h had no significant effect on the secondary and tertiary structure,

275

indicating the glycation conditions used in this work was non-denatured.

276

Theoretically, if a peptide is mono-glycated and dual-glycated by mannose, the mass will

277

increase 162.0528 Da and 324.1056 Da, respectively. One glycation site could induce an m/z shift

278

of 81.0264, 54.0176, 40.5132 and 32.4106, respectively, at 2, 3, 4 and 5 charges, accordingly.

279

Likewise, two glycation sites will double the m/z shift. As shown in Figure 4A, the m/z of

280

unglycated peptide 1-14 was 529.97453+ while the corresponding m/z of its glycated peptides were

281

583.99103+ and 638.00823+, with an m/z shift of 54.0165 and 108.0337, respectively, indicating it

282

had mono-glycated and dual-glycated peptides. Figure 4B shows the ECD MS/MS spectrum of

283

the mono-glycated peptide with m/z of 583.99103+, which exhibited a series of c and z ions

284

(c2−c13 and z2−z12). The glycation site, Lys8, was obtained by the mass difference between c7

285

and c8 ions, or between z6 and z7, which is the combined mass of lysine and mannose. Similarly,

286

the glycation sites, Leu1 and Lys8, were determined according to the ECD MS/MS spectrum of 15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

287

the dual-glycated peptides with m/z of 638.00823+ (Figure 4C). It is noted that some c and z ions

288

generated from glycated peptide may be missing due to the low intensity of ion peaks caused by

289

low collision energy. The glycation site should be determined by mass spectrum and ECD MS/MS

290

spectrum with inconsecutive or consecutive c and/or z ions that passed glycation sites. Figure 5A

291

shows that the mass spectrum of the peptide 41-60 with m/z of 771.75403+ and the mono-glycated

292

peptide with m/z of 825.77063+. The glycated peptide has an m/z shift of 54.0168, indicating that

293

one mannose molecule was added to the peptide. However, because ECD does not usually cleave

294

N-terminally to proline,32 some c and z ions, such as c7, c9, z11 and z13, were missing.

295

Similarly, other glycation sites, including Lys14, Lys 60, Lys69, Lys70, Lys75, Lys 77, Lys83,

296

Lys91, Lys100, Lys135, Lys138 and Lys141, were determined by ECD MS/MS (as shown in

297

Figure S1). As we know, the main glycation sites are lysine, arginine and N-terminus of protein.

298

There are 19 potential glycation sites in β-Lg, including one N-terminal leucine (Leu), 15 Lys and

299

3Arg. Table 2 shows the changes in glycation sites of β-Lg by ultrasound pretreatment with

300

glycation. The glycated β-Lg without ultrasound pretreatment had 12 glycated sites, including

301

Lys47, Lys60, Lys 69, Lys70, Lys75, Lys 77, Lys83, Lys91, Lys100, Lys135, Lys138 and Lys141.

302

However, 3 additional glycated sites (Leu1, Lys8 and Lys14) were found to be glycated after

303

ultrasound pretreatment. In the native state, β-Lg usually exists as a dimer through non-covalent,

304

such as hydrophobic force and electrostatic interaction.33 The formation of dimer may make some

305

glycation sites hardly react with mannose. Ultrasound pretreatment may destroy these interactions,

306

make these glycation sites exposed, and finally promote glycation. Interestingly, the result showed

307

no Arg was modified in glycated samples with and without ultrasound pretreatment. It may result

308

from the reduced freedom of Arg amine group by spatial effects from neighboring atoms.22 The 16

ACS Paragon Plus Environment

Page 16 of 40

Page 17 of 40

Journal of Agricultural and Food Chemistry

309

result conforms with previous studies, wherein they showed that glycation sites were predominant

310

Lys rather than Arg.25, 34

311

Glycation extent analysis

312

The glycation extent was estimated by both the number of glycation site and the glycation

313

extent per glycation site. It is clear that ultrasound pretreatment increased the glycation sites.

314

However, it is necessary to calculate DSP to evaluate the glycation extent of glycated β-Lg with

315

ultrasound pretreatment at different intensity. As shown in Figure 6A, the glycated β-Lg with

316

ultrasound pretreatment at 0-600 W had different glycation extent of the mono-glycated peptide

317

41-60. The relative abundance of the glycated form with m/z of 825.77063+ increased from 38.3%

318

(β-Lg-M) to the highest value of 100% (β-Lg-M-400 and β-Lg-M-600), whereas the relative

319

abundance of the unglycated form with m/z of 771.75393+ decreased from 100% (β-Lg-M) to the

320

lowest value of 25.3% (β-Lg-M-400). It suggested that ultrasound pretreatment improved the

321

glycation degree of this peptide. Another example was displayed in Figure 6B. Two mannose

322

molecules were added to the peptide 84-101, which was confirmed by the double increased m/z of

323

40.5131. The decrease in relative abundance of the ion peak with m/z of 523.53634+ and the

324

increase in relative abundance of the ion peak with m/z of 564.04944+ and 604.56254+ indicated

325

that glycated β-Lg with ultrasound pretreatment had higher glycation extent than that without

326

ultrasound pretreatment.

327

The DSP values of all glycated peptides with ultrasound pretreatment at 0-600 W were shown

328

in Figure 7. Based on the DSP value, K91 and K100 were the most reactive mannose glycation

329

sites in native β-Lg with DSP value of 0.75. After ultrasound pretreatment, its DSP value further 17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

330

increased to near 1.0. The DSP values were significantly promoted by ultrasound pretreatment as a

331

whole. The highest DSP values of glycated peptides were found at 400 W. Although some peptides,

332

1-14 and 9-40, were unglycated in the native form, they had high DSP value (≥ 0.7) after

333

ultrasound pretreatment at 400 W. It may be attributed to the exposure of these regions caused by

334

ultrasound pretreatment,10 which made some Lys residues accessible react with reducing sugar.

335

However, too high intensity ultrasound treatment may lead to the refolding of β-Lg molecules,18

336

lead to the masking of some regions, and finally cause the decrease in the glycation extent. That

337

may be the reason that β-Lg-M-600 had lower glycation degree than β-Lg-M-400.

338

Mechanism of the decrease in IgG and IgE binding of β-Lg by ultrasound pretreatment

339

combined with dry-state glycation

340

Ultrasound pretreatment coupled with glycation significantly reduced the IgG and IgE binding

341

of β-Lg, which was closely related to its structural changes. In order to explore the mechanism,

342

combined with the conventional spectrometry, FTICR-MS was applied for structural

343

characterization at the molecular level. The results above showed that the structural changes

344

were responsible for the reduction of IgG and IgE binding abilities of β-Lg.

345

As we know, the conformational epitopes play an important role in the food allergy.35 β-Lg has

346

3 conformational epitopes, in which the 15 common residues appeared mainly as an α-helix.36 The

347

secondary structure could significantly affect the IgG and IgE binding capacities. Usually, IgG and

348

IgE easily recognize and bind with the epitopes in the β-turn and random coil structures rather

349

than the α-helix and β-sheet structure.37 After glycation with mannose, the α-helix and β-sheet

350

contents of β-Lg increased, whereas the β-turn and random coil contents decreased. Therefore, the 18

ACS Paragon Plus Environment

Page 18 of 40

Page 19 of 40

Journal of Agricultural and Food Chemistry

351

IgG and IgE binding of β-Lg decreased after glycation with mannose. Moreover, the ultrasound

352

pretreatment promoted the changes, leading to the further reduction of IgG and IgE binding of

353

β-Lg.

354

Furthermore, the tertiary structure of protein determines the integrity of conformational epitopes.

355

The ultrasound pretreatment combined with glycation induced the unfolding of β-Lg, reflecting in

356

the increase in UV absorbance at 280 nm and the decrease in the intrinsic fluorescence intensity as

357

well as a red shift of λmax. The changes in the tertiary structure of β-Lg may lead to the

358

destruction of the conformational epitopes, and finally cause the reduction in IgG and IgE binding

359

abilities.

360

Besides, the hydrophobicity force is mainly responsible for maintaining the binding of antibody

361

and antigen.38 The surface hydrophobicity of glycated β-Lg was dramatically decreased, which

362

may make the IgG and IgE difficultly bind with β-Lg, and lead to the decrease of IgG and IgE

363

binding abilities. Therefore, the decrease in the surface hydrophobicity of glycated β-Lg was

364

conducive to the reduction of the IgG and IgE binding abilities. In addition, the promoted decrease

365

in the surface hydrophobicity of glycated β-Lg by ultrasound pretreatment resulted in the further

366

reduction of the IgG and IgE binding capacities.

367

Numerous studies have been done to identify the linear IgG and IgE epitopes of β-Lg. 5, 6, 36, 39-41

368

It was summarized that the IgG and IgE epitopes spread through the whole amino acid sequences

369

of β-Lg. To ensure the universality of IgG and IgE epitopes, the pooled antisera used in this study

370

were obtained from 6 rabbits and 8 CAM patients, in which the patients' age and allergy symptom

371

are different (shown in Table S1). The glycation modified or masked the IgG and IgE epitopes by

372

the covalent attachment of mannose,42 resulting in the nonrecognition of β-Lg by IgG and IgE. 19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

373

Finally, it caused the decrease in the IgG and IgE binding of β-Lg. In this case, 12 glycation sites

374

(Lys47, Lys60, Lys 69, Lys70, Lys75, Lys 77, Lys83, Lys91, Lys100, Lys135, Lys138 and

375

Lys141) were determined in the glycated β-Lg without ultrasound pretreatment (shown in Figure

376

8). The IgG and IgE were unable to recognize the epitopes due to the modification of the glycation

377

site in β-Lg molecule. Moreover, 3 additional glycation sites (Leu1, Lys8 and Lys14) were

378

identified in the glycated β-Lg with ultrasound pretreatment. It made more epitopes unrecognized

379

by IgG and IgE. Also, the DSP value showed ultrasound pretreatment promoted the glycation

380

extent. Therefore, ultrasound pretreatment promoted the reduction of the IgG and IgE binding

381

abilities by improving glycation extent.

382

In this work, it was shown that ultrasound pretreatment combined with dry-state glycation

383

significantly decreased the IgG and IgE binding of β-Lg, which was attributed to the covalent

384

binding of mannose to β-Lg and to the succeeding structural changes of the protein. Moreover,

385

ultrasound pretreatment promoted the reduction of IgG and IgE binding abilities by improving

386

glycation, reflecting in the increase of the glycation sites and DSP value. The result also

387

demonstrated that FTICR-MS was useful for analyzing the protein modifications at molecular

388

level, which is conducive to the understanding of the mechanism of the allergenicity decrease in

389

food proteins induced by processing. However, some experiments in vivo, such as double-blind

390

double-blind placebo-controlled trial (DBPC), should be measured in the future to ensure

391

reduction of allergenicity.

392

Acknowledgments

393

The authors gratefully acknowledge the financial support of the National High Technology 20

ACS Paragon Plus Environment

Page 20 of 40

Page 21 of 40

Journal of Agricultural and Food Chemistry

394

Research and Development Program of China (863 Program) (No. 2013AA102205) and National

395

Natural Science Foundation of China (NSFC) (No. 31560458).

396

References

397

1.

398

bovine milk protein glycosylation. Glycobiology 2014, 24, 220-236.

399

2.

400

to cow milk proteins in patients with milk-induced IgE-mediated and non-IgE-mediated disorders.

401

Allergy 2005, 60, 912-919.

402

3.

403

Jones, T. A.; Newcomer, M. E.; Kraulis, P. J., The structure of β-lactoglobulin and its similarity to

404

plasma retinol-binding protein. Nature 1986, 324, 383-385.

405

4.

406

bovine β-lactoglobulin adopts a β-barrel fold at pH 2. FEBS Lett. 1998, 436, 149-154.

407

5.

408

López-Fandiño, R.; Molina, E., Mapping of IgE epitopes in in vitro gastroduodenal digests of

409

β-lactoglobulin produced with human and simulated fluids. Food Res. Int. 2014, 62, 1127-1133.

410

6.

411

Epitopes on α-Lactalbumin and β-Lactoglobulin in Cow’s Milk Allergy. Int. Arch. Allergy Immunol.

412

2001, 126, 111-118.

413

7.

414

Nowak-Węgrzyn, A., Effect of heat treatment on milk and egg proteins allergenicity. Pediatr. Allergy

O'Riordan, N.; Kane, M.; Joshi, L.; Hickey, R. M., Structural and functional characteristics of

Shek, L. P. C.; Bardina, L.; Castro, R.; Sampson, H. A.; Beyer, K., Humoral and cellular responses

Papiz, M. Z.; Sawyer, L.; Eliopoulos, E. E.; North, A. C. T.; Findlay, J. B. C.; Sivaprasadarao, R.;

Fogolari, F.; Ragona, L.; Zetta, L.; Romagnoli, S.; De Kruif, K. G.; Molinari, H., Monomeric

Benedé, S.; López-Expósito, I.; Giménez, G.; Grishina, G.; Bardina, L.; Sampson, H. A.;

Järvinen, K. M.; Chatchatee, P.; Bardina, L.; Beyer, K.; Sampson, H. A., IgE and IgG Binding

Bloom, K. A.; Huang, F. R.; Bencharitiwong, R.; Bardina, L.; Ross, A.; Sampson, H. A.;

21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

415

Immunol. 2014, 25, 740-746.

416

8.

417

microfluidization at different temperatures on the antigenic response of bovine β-lactoglobulin. Eur.

418

Food Res. Technol. 2011, 233, 95-102.

419

9.

420

and potential allergenicity of the major allergen bovine β-lactoglobulin. Food Chem. 2017, 219,

421

290-296.

422

10. Stanic-Vucinic, D.; Stojadinovic, M.; Atanaskovic-Markovic, M.; Ognjenovic, J.; Grönlund, H.;

423

van Hage, M.; Lantto, R.; Sancho, A. I.; Velickovic, T. C., Structural changes and allergenic properties

424

of β-lactoglobulin upon exposure to high-intensity ultrasound. Mol. Nutr. Food Res. 2012, 56,

425

1894-1905.

426

11. Malikova, N. A.; Krzhechkovskaia, V. V.; Samenkova, N. F.; Sazhinov, G.; Marokko, I. N., Effect

427

of milk proteins and their enzymatic hydrolyzates on non-specific resistance of some systems to food

428

allergens. Vopr. Pitan. 1993, 26-30.

429

12. López-Expósito, I.; Chicón, R.; Belloque, J.; López-Fandiño, R.; Berin, M. C., In vivo methods

430

for testing allergenicity show that high hydrostatic pressure hydrolysates of β-lactoglobulin are

431

immunologically inert. J. Dairy Sci. 2012, 95, 541-548.

432

13. Zhong, J. Z.; Xu, Y. J.; Liu, W.; Liu, C. M.; Luo, S. J.; Tu, Z. C., Antigenicity and functional

433

properties of beta-lactoglobulin conjugated with fructo-oligosaccharides in relation to conformational

434

changes. J. Dairy Sci. 2013, 96, 2808-2815.

435

14. Kazem-Farzandi, N.; Taheri-Kafrani, A.; Haertlé, T., β-lactoglobulin mutation Ala86Gln improves

436

its ligand binding and reduces its immunoreactivity. Int. J. Biol. Macromol. 2015, 81, 340-348.

Zhong, J.; Liu, C.; Liu, W.; Cai, X.; Tu, Z.; Wan, J., Effect of dynamic high-pressure

Meng, X.; Bai, Y.; Gao, J.; Li, X.; Chen, H., Effects of high hydrostatic pressure on the structure

22

ACS Paragon Plus Environment

Page 22 of 40

Page 23 of 40

Journal of Agricultural and Food Chemistry

437

15. Taheri-Kafrani, A.; Tavakkoli Koupaie, N.; Haertlé, T., β-Lactoglobulin mutant Lys69Asn has

438

attenuated IgE and increased retinol binding activity. J. Biotechnol. 2015, 212, 181-188.

439

16. Verhoeckx, K. C. M.; Vissers, Y. M.; Baumert, J. L.; Faludi, R.; Feys, M.; Flanagan, S.;

440

Herouet-Guicheney, C.; Holzhauser, T.; Shimojo, R.; van der Bolt, N.; Wichers, H.; Kimber, I., Food

441

processing and allergenicity. Food Chem. Toxicol. 2015, 80, 223-240.

442

17. Huang, X.; Tu, Z.; Xiao, H.; Wang, H.; Zhang, L.; Hu, Y.; Zhang, Q.; Niu, P., Characteristics and

443

antioxidant activities of ovalbumin glycated with different saccharides under heat moisture treatment.

444

Food Res. Int. 2012, 48, 866-872.

445

18. Stanic-Vucinic, D.; Prodic, I.; Apostolovic, D.; Nikolic, M.; Cirkovic Velickovic, T., Structure and

446

antioxidant activity of β-lactoglobulin-glycoconjugates obtained by high-intensity-ultrasound-induced

447

Maillard reaction in aqueous model systems under neutral conditions. Food Chem. 2013, 138, 590-599.

448

19. Taheri-Kafrani, A.; Gaudin, J.-C.; Rabesona, H.; Nioi, C.; Agarwal, D.; Drouet, M.; Chobert,

449

J.-M.; Bordbar, A.-K.; Haertle, T., Effects of heating and glycation of β-lactoglobulin on its recognition

450

by IgE of sera from cow milk allergy patients. J. Agric. Food Chem. 2009, 57, 4974-4982.

451

20. O'Sullivan, J.; Murray, B.; Flynn, C.; Norton, I., The effect of ultrasound treatment on the

452

structural, physical and emulsifying properties of animal and vegetable proteins. Food Hydrocolloids

453

2016, 53, 141-154.

454

21. Yang, W.-H.; Tu, Z.-C.; Wang, H.; Li, X.; Tian, M., High-intensity ultrasound enhances the

455

immunoglobulin (Ig)G and IgE binding of ovalbumin. J. Sci. Food Agric. 2017, 97, 2714-2720.

456

22. Zhang, Q. T.; Tu, Z. C.; Wang, H.; Huang, X. Q.; Shi, Y.; Sha, X. M.; Xiao, H., Improved

457

glycation after ultrasonic pretreatment revealed by high-performance liquid chromatography-linear ion

458

trap/Orbitrap high-resolution mass spectrometry. J. Agric. Food Chem. 2014, 62, 2522-2530.

23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

459

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

460

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

461

capacity and conformation. Food Res. Int. 2016, 89, Part 1, 882-888.

462

24. Matulis, D.; Lovrien, R., 1-anilino-8-naphthalene sulfonate anion-protein binding depends

463

primarily on ion pair formation. Biophys. J. 1998, 74, 422-429.

464

25. Huang, X. Q.; Tu, Z. C.; Wang, H.; Zhang, Q. T.; Shi, Y.; Xiao, H., Increase of ovalbumin

465

glycation by the maillard reaction after disruption of the disulfide bridge evaluated by liquid

466

chromatography and high resolution mass spectrometry. J. Agric. Food Chem. 2013, 61, 2253-2262.

467

26. Li, Z.; Luo, Y.; Feng, L., Effects of Maillard reaction conditions on the antigenicity of

468

alpha-lactalbumin and beta-lactoglobulin in whey protein conjugated with maltose. Eur. Food Res.

469

Technol. 2011, 233, 387-394.

470

27. Matsudomi, N.; Takahashi, H.; Miyata, T., Some structural properties of ovalbumin heated at

471

80°C in the dry state. Food Res. Int. 2001, 34, 229-235.

472

28. Aoki, T.; Hiidome, Y.; Sugimoto, Y.; Ibrahim, H. R.; Kato, Y., Modification of ovalbumin with

473

oligogalacturonic acids through the Maillard reaction. Food Res. Int. 2001, 34, 127-132.

474

29. Broersen, K.; Voragen, A. G. J.; Hamer, R. J.; de Jongh, H. H. J., Glycoforms of β-lactoglobulin

475

with improved thermostability and preserved structural packing. Biotechnol. Bioeng. 2004, 86, 78-87.

476

30. Zhong, J.; Tu, Y.; Liu, W.; Xu, Y.; Liu, C.; Dun, R., Antigenicity and conformational changes of

477

β-lactoglobulin by dynamic high pressure microfluidization combining with glycation treatment. J.

478

Dairy Sci. 2014, 97, 4695-4702.

479

31. Gasymov, O. K.; Glasgow, B. J., ANS fluorescence: Potential to augment the identification of the

480

external binding sites of proteins. Biochim. Biophys. Acta, Proteins Proteomics 2007, 1774, 403-411.

24

ACS Paragon Plus Environment

Page 24 of 40

Page 25 of 40

Journal of Agricultural and Food Chemistry

481

32. Zhurov, K. O.; Fornelli, L.; Wodrich, M. D.; Laskay, U. A.; Tsybin, Y. O., Principles of electron

482

capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis.

483

Chem. Soc. Rev. 2013, 42, 5014-5030.

484

33. Barbiroli, A.; Bonomi, F.; Ferranti, P.; Fessas, D.; Nasi, A.; Rasmussen, P.; Iametti, S., Bound fatty

485

acids modulate the sensitivity of bovine β-lactoglobulin to chemical and physical denaturation. J. Agric.

486

Food Chem. 2011, 59, 5729-5737.

487

34. Huang, X. Q.; Tu, Z. C.; Wang, H.; Zhang, Q. T.; Hu, Y. M.; Zhang, L.; Niu, P. P.; Shi, Y.; Xiao,

488

H., Glycation promoted by dynamic high pressure microfluidisation pretreatment revealed by high

489

resolution mass spectrometry. Food Chem. 2013, 141, 3250-3259.

490

35. Van Regenmortel, M. H. V., What Is a B-Cell Epitope? In Epitope Mapping Protocols, Second

491

Edition, Reineke, U.; Schutkowski, M., Eds. Humana Press Inc, 999 Riverview Dr, Ste 208, Totowa, Nj

492

07512-1165 USA: 2009; Vol. 524, pp 3-20.

493

36. Li, X.; Yuan, S.; He, S.; Gao, J.; Chen, H., Identification and characterization of the antigenic site

494

(epitope) on bovine β-lactoglobulin: common residues in linear and conformational epitopes. J. Sci.

495

Food Agric. 2015, 95, 2916-2923.

496

37. Matsuo, H.; Yokooji, T.; Taogoshi, T., Common food allergens and their IgE-binding epitopes.

497

Allergol. Int. 2015, 64, 332-343.

498

38. Gooding, J. J.; Wasiowych, C.; Barnett, D.; Hibbert, D. B.; Barisci, J. N.; Wallace, G. G.,

499

Electrochemical modulation of antigen–antibody binding. Biosens. Bioelectron. 2004, 20, 260-268.

500

39. Ball, G.; Shelton, M. J.; Walsh, B. J.; Hill, D. J.; Hosking, C. S.; Howden, M. E. H., A major

501

continuous allergenic epitope of bovine β-lactoglobulin recognized by human IgE binding. Clin. Exp.

502

Allergy 1994, 24, 758-764.

25

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

503

40. Sélo; Clément; Bernard; Chatel; Créminon; Peltre; Wal, Allergy to bovine β-lactoglobulin:

504

specificity of human IgE to tryptic peptides. Clin. Exp. Allergy 1999, 29, 1055-1063.

505

41. Sélo, I.; Négroni, L.; Créminon, C.; Yvon, M.; Peltre, G.; Wal, J. M., Allergy to bovine

506

β–Lactoglobulin: Specificity of human IgE using cyanogen bromide–derived peptides. Int. Arch.

507

Allergy Immunol. 1998, 117, 20-28.

508

42. Ma, X. J.; Chen, H. B.; Gao, J. Y.; Hu, C. Q.; Li, X., Conformation affects the potential

509

allergenicity of ovalbumin after heating and glycation. Food Addit. Contam. Part A Chem. Anal.

510

Control Expo. Risk Assess. 2013, 30, 1684-1692.

511

26

ACS Paragon Plus Environment

Page 26 of 40

Page 27 of 40

Journal of Agricultural and Food Chemistry

512

Table 1. Effect of ultrasound pretreatment combined with glycation on the content (%) of β-Lg

513

secondary structures

514

samples

α-helix

β-sheet

β-turn

random coil

β-Lg

13.3 ± 0.3 a

39.6 ± 0.2a

18.5 ± 0.4 a

28.6 ± 0.5 a

β-Lg-H

12.4 ± 0.6 a

40.2 ± 0.5 a

19.2 ± 0.6 a

28.2 ± 0.4 a

β-Lg-M

18.5 ± 0.2b

39.9 ± 0.5 a

16.1 ± 0.2b

25.5 ± 0.7b

β-Lg-M-200

19.4 ± 0.3 c

40.1 ± 0.4 a

16.2 ± 0.7b

24.3 ± 0.3b

β-Lg-M-400

21.6 ± 0.6d

41.2 ± 0.3b

15.2 ± 0.4b

22.0 ± 0.4 c

β-Lg-M-600

20.2 ± 0.3 c

39.8 ± 0.5 a

16.1 ± 0.6b

23.9 ± 0.2b

Values followed by different letter (a-d) in the same column are significantly different (p < 0.05).

27

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

515

Page 28 of 40

Table 2. The glycated peptides of glycated β-Lg with ultrasound pretreated at difference intensity.

peptide location

m/z (glycated)

∆m (ppm)

sequencea

Glycation siteb

1-14

638.00823+

-4.87

(-)LIVTQTMKGLDIQK(V)

L1, K8

9-40

881.70324+

-2.27

(K)GLDIQKVAGTWYSLAMAASDISLLDAQSAPLR(V)

K14

41-60

825.77073+

-2.84

(R)VYVEELKPTPEGDLEILLQK(W)

K47

41-70

774.59065+

-3.95

(R)VYVEELKPTPEGDLEILLQKWENGEC*AQKK(I)

K60, K69

61-75

533.00504+

-3.21

(K)WENGEC*AQKKIIAEK(T)

K69, K70

71-83

446.25824+

-2.02

(K)IIAEKTKIPAVFK(I)

K75, K77

76-91

491.52284+

-2.64

(K)IPAVFKIDALNENK(V)

K83

84-101

604.56254+

-3.35

(K)IDALNENKVLVLDTDYKK(Y)

K91, K100

125-141

568.77704+

-3.43

(R)TPEVDDEALEKFDKALK(A)

K135, K138

139-148

437.91813+

-3.89

(K)ALKALPMHIR(L)

K141

516

a

C* means the alkylated cystine residue by iodoacetamide. bThe increased glycation sites after

517

ultrasound pretreatment are exhibited in bold.

28

ACS Paragon Plus Environment

Page 29 of 40

Journal of Agricultural and Food Chemistry

518 100

A

Inhibition %

80

60 1.80

7.64

11.17 13.75

1.94

18.11

40

β-Lg-N β-Lg-H β-Lg-M β-Lg-M-200 β-Lg-M-400 β-Lg-M-600

20

0 -0.5

0.0

0.5

1.0

1.5

2.0

Log(inhibitor concentration)

519 100

B

Inhibition %

80

60 2.40

7.89 11.86 14.82 20.07

2.67 40

β-Lg-N β-Lg-H β-Lg-M β-Lg-M-200 β-Lg-M-400 β-Lg-M-600

20

0 -0.5

0.0

0.5

1.0

1.5

2.0

Log(inhibitor concentration)

520 521

Figure 1. The changes of IgG (A) and IgE (B) binding abilities of β-Lg induced by ultrasound

522

pretreatment coupled with glycation. The IgG and IgE binding abilities of β-Lg samples were

523

determined by inhibition ELISA. Pooled rabbit anti-β-Lg-sera or human anti-β-Lg-sera (50 µL per

524

well) were incubated separately with 1, 5, 10, 10, 40, 48 µg mL-1 of corresponding OVA samples

525

as inhibitors.

29

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 30 of 40

[θ] (millidegree cm2 dmol-1)

15 β-Lg-N β-Lg-H β-Lg-M β-Lg-M-200 β-Lg-M-400 β-Lg-M-600

10

5

0

-5

-10 190

200

210

220

230

240

Wavelength (nm)

526 527

Figure 2. The changes in CD spectra of β-Lg induced by ultrasound pretreatment combined with

528

glycation.

30

ACS Paragon Plus Environment

Page 31 of 40

Journal of Agricultural and Food Chemistry

A

β-Lg-N β-Lg-H β-Lg-M β-Lg-M-200 β-Lg-M-400 β-Lg-M-600

0.7

Absorbance

0.6

0.5

0.4

0.3

0.2

0.1 240

260

280

300

320

340

Wavelength (nm)

529 900

β-Lg-N β-Lg-H β-Lg-M β-Lg-M-200 β-Lg-M-400 β-Lg-M-600

B

Fluorescence intensity

800 700 600 500 400 300 200 100 300

530

320

340

360

380

Wavelength (nm)

31

ACS Paragon Plus Environment

400

420

Journal of Agricultural and Food Chemistry

1900

Page 32 of 40

C

Surface hydrophobicity (H0)

1800 1700

a

a

1600

b

1500

c

1400

c d

1300 1200 1100

531

β-Lg-N

β-Lg-H

β-Lg-M

β-Lg-M-200 β-Lg-M-400 β-Lg-M-600

532

Figure 3. The changes in UV absorption (A), intrinsic fluorescence (B) spectra and surface

533

hydrophobicity (C) of β-Lg induced by ultrasound pretreatment combined with glycation. Letters

534

(a-d) in the bars mean significantly different (p < 0.05).

32

ACS Paragon Plus Environment

Page 33 of 40

Journal of Agricultural and Food Chemistry

3+ 583.9910

A

Relative Abundance %

100

AA (1-14)

80

60 3+ 529.9745 3+ 638.0082

40 530

531

∆m/z=54.0172

20 ∆m/z=54.0165

0 520

530

540

550

560

570

580

590

600

610

620

630

640

650

m/z

535 20

B

AA (1-14) 3+

L I VTQTMKGLD I QK z12 z11 z10 z9 z8 z7 z6 z5 z4 z3 z2

c4, 444.3180

0 200

400

600

800

1000

1200

m/z

536

33

ACS Paragon Plus Environment

1400

c13, 1620.8877

c12, 1492.8291

c9, 1151.6340 z9, 1179.6051

z8, 1078.5574 c8, 1094.6126

z7, 947.5170

c7, 804.4648

c5, 572.3766 z5, 600.3477 z6, 657.3692 c6, 673.4243 z+2 12, 704.8593

z4, 487.2637

5

c3, 343.2704 z3, 372.2367

10

c11, 1379.7450 z11, 1408.7114

Mannose

c10, 1264.7181 z10, 1307.6637

15

c2, 244.2020 z2, 259.1527

Relative Abundance %

FT[M+H] =583.9910 m/z

c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13

1600

1800

Journal of Agricultural and Food Chemistry

15

C

AA (1-14) c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13

FT[M+H]3+=638.0082 m/z

L I VTQTMKGLD I QK

0 200

400

800

1000

1200

1400

c12, 1654.8819

z12, 1507.7798 c11, 1541.7978

c10, 1426.7709 z11, 1408.7114

c8, 1256.6653 z10, 1307.6637 c9, 1313..6868

Mannose

z9, 1179.6051

z8, 1078.5574

Mannose

z7, 947.5170 c7, 966.5176

z+2 13, 1507.7798

c6, 835.4771

600

c5, 734.4294

z5, 600.3477 c4, 606.3708 z6, 657.3692

z4, 487.2637 c3, 505.3232

z3, 372.2367 c2, 406.2548

5

z2, 259.1527

10

c+2 13, 891.9620

z13 z12 z11 z10 z9 z8 z7 z6 z5 z4 z3 z2

c1, 293.1707

Relative Aboundance %

Page 34 of 40

1600

1800

m/z

537 538

Figure 4. Mass spectrum of the peptide 1-14 (A) and ECD MS/MS spectra of the glycated peptide

539

1-14 with m/z of 583.99103+ (B) and 638.00823+ (C) from the glycated β-Lg after ultrasound

540

pretreatment at 400 W. The m/z differences are indicated with numbers and arrows. The sequence

541

of peptide is shown on the top of figure. The determined glycation site (blue bold) is shown by an

542

arrow with mannose. The c and z ions are indicated by the number.

34

ACS Paragon Plus Environment

Page 35 of 40

Journal of Agricultural and Food Chemistry

Relative Abundance %

120

A

AA (41-60) 3+ 825.7706

100

80

60

40

3+ 771.7538

20

∆m/z=54.0168

0 750

760

770

780

790

800

810

830

840

850

m/z

543

20

B

AA (41-60) c10c11c12c13c14c15c16

c8

VYVEELKPTPEGDLEILLQK

3+ 15 FT[M+H] =825.7707 m/z

z15 z14

z10 z9 z8 z7 z6 z5 z4 z3 z2

z12

Mannose

0 200

400

600

800

1000

1200

1400

1600

1800

c16, 1991.9867

z15, 1840.0073 c15, 1878.9005

z14, 1726.9134 c14, 1749.8570

c13, 1636.7741

c12, 1521.7559

c11, 1464.7324

c10, 1335.7042 z12, 1339.7125

c8, 1173.5964 z10, 1141.6129

z8, 955.5525

z9, 1012.5746

z7, 840.5276

z6, 727.4442 c6, 750.3996

z5, 598.4027 c5 637.3167

z2, 259.1523

5

z4, 485.3191 c4, 508.2745

10

z3, 372.2356

Relative intensity %

c4 c5 c6

544

820

2000

m/z

545

Figure 5. Mass spectrum of the peptide 41-60 (A) and ECD MS/MS spectrum of the glycated

546

peptide 41-60 with m/z of 825.77073+ (B) from the glycated β-Lg after ultrasound pretreatment at

547

400 W. The m/z differences are indicated with numbers and arrows. The sequence of peptide is 35

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

548

shown on the top of figure. The determined glycation site (blue bold) is shown by an arrow with

549

mannose. The c and z ions are indicated by the number.

36

ACS Paragon Plus Environment

Page 36 of 40

Page 37 of 40

Journal of Agricultural and Food Chemistry

A

B

AA (41-60)

AA (84-101) 4+

3+

50

0

Relative Abundance %

771.7539

100

825.7706

760

780

800

820

840

3+

100

3+

771.7540

825.7707

50

0

760

780

800

820

4+ 4+

523.5363

604.5625

50

0

520

540

560

580

Relative Abundance %

Relative Abundance %

3+

771.7538

0

760

780

800

820

4+

4+

50

604.5623

523.5362

520

540

560

580

840

Relative Abundance %

Relative Abundance %

550

3+

50

780

800

820

640

50

4+

564.0493 4+

523.5364

0 500

520

540

560

580

600

620

640

564.0494

825.7704

760

620

4+

771.7537

0

600

604.5625

100

3+

100

640

4+

825.7706

50

620

4+

3+

100

600

564.0492

100

0 500

840

564.0494

100

500

Relative Abundance %

Relative Abundance %

Relative Abundance %

3+

840

100

4+

604.5624

50 4+

523.5362

0 500

520

540

m/z

560

580

600

620

640

m/z

551

Figure 6. Mass spectra of the glycated peptides 41-60 (A) and 84-101 (B). The red, blue, magenta

552

and green spectra refer the peptides from glycated β-Lg with ultrasound pretreatment at 0, 200,

553

400 and 600 W, respectively.

37

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 38 of 40

β-Lg-M β-Lg-M-200 β-Lg-M-400 β-Lg-M-600

1.2 b

b

1.0 c

c

b

DSP

c

c

c

b,c a a

a

b

b

b

b

b

a a

c

b

b,c

b a a

a a

0.4

a a

0.2

0.0

b,c

d

c

a 0.6

b,c

c

0.8

a,b

c

c

1-14

9-40

41-60

41-70

61-75

71-83

76-91

84-101 125-141 139-148

Peptide

554 555

Figure 7. DSP values of the glycated peptides from the glycated β-Lg with ultrasound

556

pretreatment at 0, 200, 400 and 600 W. Letters (a-d) in the bars mean significantly different (p