Improved Antioxidant Activity and Glycation of Î±-Lactalbumin after

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Improved antioxidant activity and glycation of #-lactalbumin after ultrasonic pretreatment revealed by high-resolution mass spectrometry Jun Liu, Zong-cai Tu, Yan-hong Shao, Hui Wang, Guang-xian Liu, Xiao-mei Sha, Lu Zhang, and Ping Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03920 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 6, 2017

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Journal of Agricultural and Food Chemistry

Improved antioxidant activity and glycation of α-lactalbumin after ultrasonic pretreatment revealed by high-resolution mass spectrometry

1 2 3 4 5 6 7 8

Jun Liu*, Zong-cai Tu*, †, ‡, Yan-hong Shao*, Hui Wang†, ‡, Guang-xian Liu*, Xiao-mei

9

Sha*, Lu Zhang*, Ping Yang*

10 11 12 13

*

14

Normal University, Nanchang, Jiangxi 330022, China;

15

†

16

Nanchang, Jiangxi 330047, China;

College of Chemistry and Chemical Engineering, College of Life Science, Jiangxi

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

17 18 19

‡

20

E-mail:

21

(Hui-Wang)

Corresponding authors. Tel.: +86-791-8812-1868; fax: +86-791-8830-5938. [email protected]

(Zong-cai

Tu),

[email protected]

22 23 24 25 26 27 28 29 30

ACS Paragon Plus Environment


31

Abstract: High-resolution mass spectrometry was performed to investigate the

32

relationship between bovine α-lactalbumin (α-LA) subjected to ultrasonication and

33

glycation treatment with respect to antioxidant activity. After α-LA was pretreated by

34

ultrasonication combined with glycation, the treated α-LA showed low intrinsic

35

fluorescence emission and high antioxidant activity at increased ultrasonic power

36

levels. Prior to ultrasonic pretreatment, three glycated sites were identified, whereas

37

the number of glycation sites was increased to four, four, five and six after ultrasonic

38

power at 60, 90, 120, and 150 W/cm2, respectively, for 15 min. Thus, no obvious

39

difference was found among the glycation sites at the ultrasonic power of 60 and 90

40

W/cm2. The average degree of substitution per peptide molecule of α-LA was used to

41

evaluate the glycation level for per glycation site. All the samples pretreated by

42

ultrasonication exhibited a higher glycation level compared with the untreated

43

samples. Ultrasonic power at 150 W/cm2 showed the most highly enhanced glycation

44

extent and antioxidant activity. Therefore, the intensified glycation extent and the

45

conformational changes of protein were responsible for the increase of antioxidant

46

activity of α-LA. Moreover, high-resolution mass spectrometry is an efficient

47

technique to understand the mechanism of the improved antioxidant activity.

48 49 50

Keywords: α-lactalbumin, ultrasonication, glycation, antioxidant activity, mass

51

spectrometry

52 53 54 55


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ABBREVIATION USED

57

α-LA, alpha-lactalbumin; Glc, glucose; DSP, the average degree of substitution per

58

peptide molecule; MRPs, Maillard reaction products; PBS, phosphate buffer solution;

59

AA, amino acid; TFA, trifluoroacetic acid; DTT, DL-dithiothreitol; ABTS,

60

2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate);

61

preparation; SEC, size exclusion chromatography; HPLC, high performance liquid

62

chromatography; ETD MS/MS, electron transfer dissociation mass spectrometry/mass

63

spectrometry.

FASP,

64 65 66 67 68 69 70 71 72 73 74 75 76 77


filter-aided

sample


78 79

Introduction

80

Small bovine protein α-lactalbumin (α-LA), is suited as an ingredient for infant

81

nutrition in the food1, which consists of 123 amino acid residues and four disulfide

82

bridges, and has molecular weight around 14.2 KDa2. α-LA is a simple model Ca2+

83

binding protein and has immune-modulating3, antioxidant4, antibacterial5 or antitumor

84

activity6, 7. Its hydrolyzate also exhibit antioxidant activity8. Reported strategies, such

85

as heat treatment9, 10, glycation11 and high-intensity ultrasonic treatment12 nationally

86

modulate α-LA functionality and thus address physical and chemical pathways. For

87

these processes, glycation is the first step of Maillard reaction occurring between

88

amino and carbonyl compounds, and plays an important role in food processing,

89

especially in protein modification13. Moreover, α-LA is supplemented to infant

90

formulae which undergo the glycation reaction, then may modulate its functionality14.

91

The weight ratio of protein to sugar, reaction temperature and time; pH, water activity;

92

and reactant structure are the major factors that influence the glycation reaction; these

93

factors affect the glycation reaction between α-LA and maltose and reduces the

94

antigenicity of α-LA15. In addition, Velusamy et al. studied the influence of glucose

95

and pH on the glycation of α-LA and found that glucose and pH affect the

96

stabilization of α-LA16. Although glycation is widely used to improve functional

97

properties of α-LA13, 17, a single modification alone cannot improve the functional

98

properties to a satisfactory result.

99

High-intensity ultrasonication, an emerging nonthermal technology, presents a


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great deal of successful improvements on the functional properties of food in four

101

ways, including heating effects, acoustic cavitation, acoustic streaming, and fluid

102

particle oscillations18,

103

glycation of bovine serum albumin (BSA)20 and unfold the structure of ovalbumin21.

104

Ultrasonication and glycation can disrupt the secondary and tertiary structure of

105

α-LA22, and glycation can greatly improve the antioxidant activity8. Glycation occurs

106

on the Lys and Arg of a protein and alters the peptides. Thus, the conformational

107

changes of α-LA owing to ultrasonic pretreatment coupled with glycation were

108

associated with the antioxidant activity. Moreover, the glycation extent is also

109

important for the explanation of the functional properties of glycoprotein. Glycation

110

extent was evaluated mostly using traditional methods and parameters, such as free

111

amino acid content, browning intensity and fluorescence intensity etc15, 23. However,

112

in these methods, the glycation sites and glycation extent at each site in the protein

113

cannot be identified because the change in glycation degree are determined at the

114

protein level. Mass spectrometry is widely used to analyze the nature and extent of

115

protein modification24, 25, and exactly investigate the processes inside the protein. To

116

date, although the mechanisms by which proteins and Maillard conjugates exert their

117

antioxidant activity are poorly understood26, the influence of ultrasonic pretreatment

118

combined with glycation on the antioxidant activity and glycation extent of α-LA has

119

not been studied. Providing a method of protein modification in the food industry also

120

requires investigating its antioxidant activity.

121

19

. In our previous work, ultrasonication can promote the

The overall goal of this research was to study the impact of ultrasonic power on



122

the antioxidant activity and glycation extent of α-LA treated by glycation. We firstly

123

applied the ultrasonic technique to the structural perturbation of α-LA. Ultrasonicated

124

α-LA was then subjected to glycation. The antioxidant activity of treated α-LA was

125

evaluated by the ABTS radical-scavenging activity. The glycation sites and extent of

126

α-LA were further examined by high-resolution mass spectrometry. The results of this

127

research enhance our understanding of the relationship between antioxidant activity

128

and structural changes of α-LA induced by ultrasonic pretreatment combined with

129

glycation.

130

Materials and methods

131

Chemicals and materials

132

Alpha-lactalbumin (α-LA) from bovine milk (L6010, Type Ⅲ, ≥ 85%), Glucose

133

(G8270), pepsin (P6887, 3,200-4,500 units/mg protein) were purchased from Sigma

134

Chemical Co. (St. Louis, MO, USA); DL-1,4-Dithiothreitol (AC165680050) was

135

purchased from Fisher Scientific Inc. (Waltham, MA, USA). All other reagents and

136

solvents used were of analytical and high performance liquid chromatography (HPLC)

137

grade.

138

Sample Preparation

139

Native α-LA (100 mg) was dissolved in 100 mL of 50 mM phosphate buffer saline

140

(PBS) at pH 7.4. Ten milliliters of α-LA were split into 25 mL flat bottom conical

141

flasks, and immersed in ice bath. The solution was treated by probe sonicator

142

(Misonix Qsonica Q700 Sonicator, USA, 20 kHz) equipped with a microtip probe

143

(1/8 in. = 3 mm) with a 9s on and 1s off pulsation at an actual ultrasonic intensity of 0,


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60, 90, 120 and 150 W/cm2 for 15 min, respectively, to ensure the temperature of the

145

sample solution is not elevated (lower than 15 oC).

146

In case of glycation, 1.0 mg of glucose (Glc) was added to 1.0 mL of the native

147

α-LA and ultrasonicated α-LA solution, separately. After lyophilization, the powders

148

of native BSA, native α-LA-glucose, ultrasonicated α-LA-glucose complex were

149

incubated at 65% relative humidity (saturated potassium iodide solution) and 55 oC

150

for 3 h. The reaction was stopped by transferring the sample tubes into an ice bath.

151

The free glucose and salts were filtered by a Centricon centrifugal filter unit (3000 Da

152

cutoffs, Millipore, Bedford, MA, USA). The concentration of all the samples were

153

adjusted to 10 mg/mL and stored at 4 oC for further analyses. Native α-LA was named

154

N-LA. The glycated samples with ultrasonic pretreatment at 0, 60, 90, 120, and 150

155

W/cm2 for 15 min were named N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120,

156

LA-Glc-150, respectively. The treatments were performed in triplicates.

157

Size exclusion chromatography

158

Sample solution (30 uL) were purified by size exclusion chromatography (SEC) on a

159

TSK Gel 3000 SWXL column (TOSOH Bioscience, King of Prussia, PA, USA) using

160

an Agilent 1100 (Agilent Technologies, Palo Alto, CA, USA) HPLC system. The

161

separation was performed with 0.1 M ammonium acetate (pH 6.8) at a flow rate of 0.5

162

mL/min and monitored with 280 nm ultraviolet (UV) detection.

163

Intrinsic fluorescence emission spectroscopy



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1.0 mg/mL of samples were prepared with PBS (50 mM, pH 7.4). The intrinsic

165

fluorescence emission spectra were obtained by a Hitachi F-7000 fluorescence

166

spectrophotometer (Hitachi, Ltd, Tokyo, Japan). The emission spectra were recorded

167

from 300 nm to 400 nm (both at a constant slit of 5 nm) with excitation at 290 nm and

168

PBS was used as blank.

169

Determination of ABTS+ radical scavenging activity

170

The ABTS+ radical scavenging activity was estimated using a previously reported

171

method27. The ABTS+ was formed by adding K2S2O8 to ABTS. In brief, 20 µL of each

172

protein sample (2, 4, 6, 8, and 10 mg/mL) was mixed with the diluted ABTS+ solution

173

(absorbance of 0.70 ± 0.01 at 734 nm). The mixture was incubated in the dark for 6

174

min, and the absorbance of mixture was measured at 734 nm using a SynergyTM HT

175

Multi-Mode Microplate Reader (BioTek Instruments Co. Ltd., VT, USA). The ABTS+

176

radical scavenging activity (%) was calculated:

177

ABTS radical scavenging activity (%) =

!"#

× 100%

178

where Acontrol is the absorbance of the control (ABTS+ solution without samples) and

179

Asample is the absorbance of the samples.

180

Filter-aided sample preparation (FASP)

181

Protein digestion was conducted using to the FASP method according to the method

182

of Chen et al.25 with slight modifications.10 µL samples (10 mg/mL) were mixed with

183

200 µL of 150 mM urea (UA) buffer (8 M urea, 150 mM Tris-HCl pH 8.0) and 20 µL

184

DTT (100 mM) and incubated at 95 oC for 5 min, then cooled in the ice bath. The

185

mixture was transferred to a Centricon centrifugal filter unit (10000 Da cutoffs,


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Millipore, Bedford, MA, USA). The samples were centrifuged at 14000 × g for 15

187

min at 25 oC, 200 µL UA buffer was then added and centrifuged at 14000 × g for 15

188

min. After, 100 µL iodoacetamide (50 mM) in UA buffer was added and incubated at

189

room temperature in the dark for 60 min, and then centrifuged at 14000 × g for 10

190

min. The filters were washed thrice using UA buffer (100 µL), followed by the

191

additional of 100 µL ammonium bicarbonate solution (50 mM, pH 8.5), then

192

centrifuged at 14000 × g for 10 min and repeated this step thrice. 30 µL pepsin (10

193

mg/mL) was added to each filter and incubated 4 oC for 15 min. The reaction was

194

then quenched by adding 6 µL of 10% TFA. The filtered solution was collected by

195

centrifugation at 14000 × g for 15 min for next step analysis.

196

Analysis by HPLC-ETD-MS/MS

197

Samples were analyzed on a Thermo Fisher Q Exactive Mass Spectrometer (Thermo

198

scientific, Waltham, MA, USA). Solvent A was 0.1% formic acid (FA) in ultrapure

199

H2O, whereas solvent B consisted of 84% acetonitrile in H2O, 0.1% FA. For analysis

200

150 µL of proteolytic peptides, the digested sample was injected onto a 75 µm i.d. ×

201

100 mm 3µm-C18 column (EASY-column, Thermo scientific, Waltham, MA, USA).

202

After desalting for 20 min with A, the peptides were eluted at 300 nL/min with a

203

gradient of 4-50% B for 40 min, 50-100% B for 5 min, and 100% B for 1 min, then

204

analyzed using a Thermo Fisher Q Exactive Mass Spectrometer. Detection mode:

205

positive ions. The scan range was set to 150–2000 m/z, and a resolution of 70000 at

206

m/z 200 was applied for acquiring survey scans. The dry gas was set to a flow of 7.0

207

L/min, a capillary temperature of 250 °C, and a spray voltage of 1.8 KV. The m/z of



208

peptides and peptide fragments was detected by the following methods: full

209

scan-every time, and then collected 20 fragmentography (MS2 scan).

210 211

To further compare the extent of glycation extent of each peptide, the average degree of substitution per peptide molecule (DSP) of α-LA was calculated: DSP =

∑7.89 i × I+,+-./,0 . × 123456, ∑7.89 I+,+-./,0 . × 123456,

212

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

213

glucose units attached to the peptide in each glycated form.

214

Database Search

215

Original RAW file was performed to Mascot Server (Matrix Science) by proteome

216

discoverer, and ETD was used as the method of check database. The following search

217

parameters were used: enzyme = no specific, missed cleavage = 5, fixed modification:

218

carbamidomethyl (C), variable modification: oxidation (M), glycation (K, R and N).

219

Mass values: monoisotopic peptide mass tolerance = ± 10 ppm, MS/MS tolerance = ±

220

0.5 Da. The following search database were used: > sp|P00711|LALBA_BOVIN

221

Alpha-lactalbumin OS = Bos taurus GN = LALBA PE = 1 SV = 2

222

MMSFVSLLLVGILFHATQAEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTS

223

GYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTD

224

DIMCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL. STQTΑLA. Parent ion

225

tolerance = ± 10 ppm, daughter ion tolerance = ± 0.5 Da. Results of filtering

226

parameters: Mascot Score ≥ 20.

227

Results and Discussion


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228

Size exclusion chromatography

229

Size exclusion chromatography (SEC) separates biomolecules according to their

230

molecular size28. Fig. 1 displays SEC diagrams for N-LA, N-LA-Glc, LA-Glc-60,

231

LA-Glc-90, LA-Glc-120, and LA-Glc-150. Monomer of N-LA was calculated by SEC,

232

after glycation; the monomer of α-LA-Glc conjugates was also observed. However,

233

the elution time (monomer) of ultrasonicated α-LA-Glc conjugates shifted to around

234

19.5 min, which is much shorter than the elution time of N-LA and N-LA-Glc at 20.6

235

min and 19.8 min, respectively. This result indicates the formation of proteinaceous

236

high molecular weight components after ultrasonication combined with glycation.

237

However, no obvious difference is found in the α-LA-Glc conjugates pretreated by

238

different ultrasonic power levels. This finding may be that the SEC could not reveal

239

their changes exactly and further work needs to be done through mass spectrometry.

240

On the other hand, the aggregation of N-LA was also assessed by SEC (Fig. 1). The

241

aggregation of N-LA was not observed at 65% relative humidity (saturated potassium

242

iodide solution) and 55 oC for 3 h, indicating that α-LA was not denatured at current

243

glycation conditions.

244

Intrinsic fluorescence emission spectroscopy

245

The intrinsic tryptophan fluorescence emission spectra of all the samples were

246

presented in Fig. 2. When excited at 290 nm, the intensity of N-LA, N-LA-Glc,

247

LA-Glc-60, LA-Glc-90, LA-Glc-120, and LA-Glc-150 were gradually reduced,

248

particularly for LA-Glc-150, which indicates the conformational structure of α-LA



249

was dramatically changed by ultrasonic pretreatment combined with glycation, this

250

result can be attributed to the fact that the relatively increased exposure of Trp

251

residues in more hydrophilic surroundings29, solvent relaxation30 and more quenching

252

agents were produced in the conjugates. Furthermore, it may result from the shielding

253

effect of the carbohydrate bound to α-LA31. The α-LA undergoes conformational

254

changes around the Trp residues due to the heating treatment32, as this study used dry

255

heating for glycation. More importantly, the Trp/Tyr residues has the ability to donate

256

a proton. This result may have resulted in their having different antioxidant activities.

257

Antioxidant activity

258

As shown in the Fig. 3, the ABTS radical-scavenging activity of α-LA significantly

259

improved after glycation to a greater degree compared with that of N-LA (p < 0.05).

260

The enhancement may be due to the reduction of the radical cation because of the

261

reaction between the ABTS radicals and Maillard reaction products (MRPs)27.

262

Furthermore, α-LA undergoes conformational changes around the Trp/Tyr residues

263

because of ultrasonication and glycation (Fig. 2). Antioxidant milk-derived peptides

264

are composed of 5 to 11 amino acids, which include the hydrophobic amino acids Pro,

265

His, Tyr, and Trp14, 33. However, no obvious difference existed between the ABTS

266

radical-scavenging activity of N-LA-Glc and LA-Glc-60 (p > 0.05), but the activity of

267

N-LA-Glc, LA-Glc-90, LA-Glc-120 and LA-Glc-150 were significantly different

268

from one another in (p < 0.05). This finding can be explained by the fact that the

269

compact structure of α-LA could be effectively unfolded at increased ultrasonic

270

power12, thereby accelerating the glycation between α-LA and glucose, producing


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271

more MRPs, and finally improving the antioxidant activity.

272

Glycation Site Determination

273

It is known that α-LA has 12 Lys and 1 Arg, including Lys5, Lys13, Lys16, Lys58,

274

Lys62, Lys79, Lys93, Lys94, Lys98, Lys108, Lys114, Lys122 and Arg10, and the

275

N-terminal of Asn45 was linked with a N-acetylglucosamine. Therefore, α-LA

276

contains at least 13 potential glycated sites. Theoretically, if a peptide was

277

mono-glycated by glucose, the corresponding m/z of peaks with 1, 2, 3, 4, or 5

278

charges will display a mass increase of 162.0528 Da, with m/z change of 162.0528,

279

81.0264, 54.0176, 40.5132, and 32.4106 Da, respectively. For the dual-glycated

280

peptides, the mass increase will be 324.1056 Da.

281

As shown in the figure 4, the m/z peaks of the unglycated peptide 61-71, 91-102,

282

104-115, 105-119, and 111-123 were 434.51073+, 462.92253+, 485.93093+, 604.65383+,

283

570.29263+ Da, while the corresponding m/z of glycated peptides were 488.52813+,

284

570.97853+, 539.94843+, 658.67163+and 624.29033+ Da, respectively. The m/z shift of

285

these peaks were 54.0174, 108.056, 54.0175, 54.0178, and 53.9977 Da, respectively,

286

indicating that all these peptides had mono-glycated or dual-glycated peptides.

287

A detailed map of the glycated sites of α-LA was obtained using the ETD

288

MS/MS. The determination of glycated sites Lys62, Lys94, Lys98, Lys108, Lys114

289

and Lys122 by ETD MS/MS is depicted in Fig. 5. The ETD MS/MS spectrum of the

290

mono-glycated

291

488.52813+ exhibited a series of c and z ions (c2−c11 and z2−z11). The glycation site,

292

Lys62, was obtained by the mass difference between the c1 and c3 ions, or between

peptide

61

C(carbamidomethyl)KDDQNPHSSN71


with

m/z

of


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293

the z9 and z11, which is the combined mass of Lys and glucose. The glycation sites,

294

Lys98, Lys108, Lys114 and Lys122 were determined according to the ETD MS/MS

295

spectrum of the mono-glycated peptides with m/z values of 428.73784+, 539.94843+,

296

658.67163+, 624.29033+ respectively. A series of c and z ions in the Fig. 5C, D, E, F

297

was

298

91

299

104

300

105

LAHKALC(carbamidomethyl)SEKLDQWL119,

301

111

C(carbamidomethyl)SEKLDQWLC(carbamidomethyl)EKL123

302

Similarly, Fig. 5B shows the mass spectrum of the fragmentation of dual-glycated

303

peptides

304

glycation sites were identified to be Lys94 and Lys98.

detected,

and

matched

well

with

the

fragments

of

peptides

C(carbamidomethyl)VKKILDKVGINY103, WLAHKALC(carbamidomethyl)SEKL115,

91

respectively.

C(carbamidomethyl)VKKILDKVGIN102 with m/z of 570.97853+. The

305

Table 1 shows that the sequence and glycation sites of N-LA-Glc containing

306

three glycation sites, which include Lys62, Lys94, Lys98. After α-LA was pretreated

307

by ultrasonic power at 60 and 90 W/cm2 for 15 min, four sites (Lys62, Lys94, Lys98,

308

Lys108) were glycated. When the sample was ultrasonicated at 120 W/cm2 for 15 min,

309

five sites (Lys62, Lys94, Lys98, Lys108, Lys114) were glycated. Furthermore, six

310

sites (Lys62, Lys94, Lys98, Lys108, Lys114 and Lys122) were found to be glycated

311

in the sample ultrasonicated at 150 W/cm2 for 15 min. The increase of the glycated

312

sites suggested that the α-LA structures loosened under ultrasonic power, which

313

facilitated the glycation reaction and exposed more reactive sites. In this study,

314

Lys114 and Lys122 were not observed in LA-Glc-60 and LA-Glc-90, but they were


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detected in the LA-Glc-120 and LA-Glc-150, suggesting that Lys114 and Lys122

316

were glycated with Glc. The glycation proceeded at ultrasonic power levels of 120

317

and 150 W/cm2, which enabled the unfolding of α-LA structure, particularly that of

318

LA-Glc-150. Meanwhile, most glycation sites were measured. High ultrasonic power

319

levels can perturb the protein conformation to a considerably high extent, leading to

320

the intensified glycation. Fig. 6 showed that three additional glycation sites, Lys108,

321

Lys114 and Lys122 were found in the glycated α-LA after ultrasonic pretreatment.

322

Interestingly, the results were consistent with those obtained in our previous studies

323

where the glycated samples with and without ultrasonic pretreatment predominantly

324

occurred on Lys but not on Arg20. Thus, ultrasonic pretreatment can increase the

325

glycation sites by the conformational changes of α-LA.

326

Effects of ultrasonication on the glycation extent of α-LA

327

When α-LA was treated by ultrasonication, the glycation site was significantly

328

increased (Table. 1), indicating that ultrasonication can effectively improve the

329

glycation extent between α-LA and glucose. The DSP values of the glycated peptides,

330

which included 61-71, 91-102, 104-115, 105-119 and 111-123 with ultrasound

331

pretreatment at 0–150 W/cm2, are shown in Fig. 7. The DSP values were significantly

332

promoted by ultrasound pretreatment as a whole. For example, the DSP value of

333

Lys62 was 0.5 in N-LA-Glc. After the sample ultrasonicated at 60–150 W/cm2 for 15

334

min, its DSP further increased to 0.76, 0.79, 0.87 and 0.92. The highest DSP values of

335

all the glycated peptides were found at 150 W/cm2. The peptide, 104-115 was

336

glycated in LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150, its DSP value was



337

gradually increased with the increase of ultrasonic power. When ultrasonic power

338

reached to 150 W/cm2, glycated peptide105-119 also had a higher DSP value than

339

LA-Glc-120. Thus, ultrasonication caused the exposure of these regions22, with the

340

more ultrasonic power, the regions will have a better exposure, which then accelerated

341

the glycation and improved the glycation extent of α-LA. This result agreed with the

342

previously performed a similar study on BSA glycation under the influence of

343

ultrasonication20.

344

Mechanism of the increase in the antioxidant activity of α-LA by ultrasonic

345

pretreatment combined with glycation.

346

The extent of Maillard reaction is important for the explanation of the functional

347

behaviors of MRPs. In this study, ultrasonic pretreatment combined with glycation

348

significantly improved the antioxidant activity of α-LA, which was closely related to

349

its structural changes. To explore the mechanism, high resolution mass spectrometry

350

was applied for structural characterization at the molecular level. The results above

351

show that the structural changes were responsible for the increase of antioxidant

352

activity of α-LA.

353

Peptides possessing antioxidant activity are generally small in size, with

354

molecular weight not exceeding 3 kDa34 and some amino acid residues also possess

355

antioxidant activity, especially Cys, Trp, Tyr and Met35. Also, lien et al. reported that

356

Cys is an important element of the antioxidant system of the neonate36. The

357

hydrophobic amino acids Pro, His, Tyr, and Trp can serve as hydrogen donors33, thus

358

stabilizing free radicals thus stabilizing free radicals and accounting for protein’s


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359

inherent ability to act as an antioxidant. The structure of α-LA contains four Phe, four

360

Trp and four Tyr (Phe9, Phe31, Phe53, Phe80, Trp26, Trp60, Trp104, Trp118, Tyr18,

361

Tyr36, Tyr50 and Tyr103), which increase its potential to produce antioxidative

362

peptides. According to Leïla Sadat et al., the peptides 21VSLPEW26, 36YDTQA40,

363

101

364

scavenging capacity and contained at least one Tyr/Trp residue8. This finding can be

365

explained by the quenching of the Tyr-phenolic and Trp-indolic hydrogen (H+) by the

366

ABTS+ radicals to form more stable phenoxyl and indolyl radicals8, 37. The intrinsic

367

fluorescence spectra of all the samples decreased compared with that of N-LA in the

368

present work (Fig. 2), this result indicated that ultrasonic pretreatment combined with

369

glycation significantly affects the conformation of proteins changes around Tyr/Trp

370

residues38, 39. Moreover, during glycation a relevant variation in protein charge occurs

371

due to the involvement of the basic amino groups of proteins in Maillard-type

372

reaction40, this modification may induce the change of antioxidant activity. Therefore,

373

the antioxidant activity of α-LA increased after glycation with glucose.

INY103, 101INYW104 and 115LDQW118 possesses remarkable ABTS radical

In addition, the peptide 91CVKKILDKVGINY103 of all the samples contained the

374 375

antioxidant peptide 100INY103. After glycation, Cys91, Lys94 and Lys98 were

376

acetylated and glycated separately that led to the conformation of the peptide changes

377

around Cys91and Tyr103 residues, suggesting that the peptidic bond or structural

378

peptide conformation enhanced the antioxidant activity of the constitutive peptide and

379

amino acids37. However, four additional glycated peptides (104WLAHKALCSEKL115,

380

105

LAHKALCSEKLDQWL119, 105LAHKALCSEKLDQWL119 and



381

111

382

was glycated, the conformation of the peptide 104-115 around Cys111 and Trp104

383

was changed, suggesting that the change of the structural peptide conformation

384

enhanced the antioxidant activity of the constitutive peptide. In the sample

385

ultrasonicated at 120 W/cm2 for 15 min, the glycated peptide 105-119 was identified

386

that includes antioxidant peptide 115LDQW118, the conformation of the peptide around

387

His107, Cys111, and Trp118 residues was similarly changed. Moreover, the peptides

388

105-119 and 111-123 were glycated, the same phenomenon around His107, Cys111,

389

Trp118 and Cys120 residues was observed to have actual change in sample

390

ultrasonicated at 150 W/cm2 for 15 min, and had finally improved antioxidant activity

391

of α-LA. The antioxidant activity of the increasing ultrasonic power levels was

392

observed in the order 150 W/cm2 > 120 W/cm2 > 90 W/cm2 > 0 W/cm2. Also, the DSP

393

value showed ultrasonic pretreatment promoted the glycation extent (Fig. 7).

394

Therefore, ultrasonic pretreatment increased the antioxidant activity of α-LA by

395

altering the conformation of α-LA and improving its glycation extent.

396

CSEKLDQWLCEKL123) were found after ultrasonic pretreatment. When Lys108

In summary, the results of the experiment demonstrated that ultrasonication

397

combined with glycation significantly improved the glycation and antioxidant

398

activities of α-LA. This finding was attributed to the covalent binding of glucose to

399

α-LA and to the succeeding structural changes of α-LA around some amino acid

400

residues. Moreover, ultrasonic pretreatment promoted the increase of antioxidant

401

activities by improving glycation. The result was reflected by the reduction in the

402

intrinsic fluorescence emissions and increase in the glycation sites and DSP value. It


Page 18 of 36

Page 19 of 36


403

also demonstrated that high-resolution mass spectrometry is a powerful tool for

404

analyzing the protein modifications at a molecular level, thereby enabling the

405

exploration of the mechanism of improved antioxidant activity in food proteins during

406

food processing. Therefore, ultrasonication combined with glycation was revealed as

407

a good technology for improved antioxidant activity of proteins.

408

Acknowledgements

409

This work was supported by Chinese National Natural Science Foundation (No.

410

31360374), the earmarked fund for China Agriculture Research System (CARS-45),

411

Chinese National Natural Science Foundation (No. 31460395), and Excellent Youth

412

Foundation of Jiangxi Province (20162BCB23017).

413

References

414

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415

β-lactoglobulin and α-lactalbumin—Technological implications for processing. Int.

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(3) Meng, X.; Li, X.; Wang, X.; Gao, J.; Yang, H.; Chen, H., Potential allergenicity

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response to structural modification of irradiated bovine α-lactalbumin. Food Funct.

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2016, 7(7), 3102-3110.

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rheological properties of peroxidase-cross-linked apo-α-lactalbumin after heat

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characteristics

and

emulsifying


properties

of

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(13) Ter Haar, R.; Westphal, Y.; Wierenga, P. A.; Schols, H. A.; Gruppen, H.,

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digestive proteolysis. Food Funct. 2015, 6(4), 1229-1240.

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maltose. Eur. Food Res. Technol. 2011, 233(3), 387-394.

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α-lactalbumin. Food Biophys. 2016, 11(1), 108-115.

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Food Chem. 2014, 62(12), 2522-2530.

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enhances the immunoglobulin (Ig) G and IgE binding of ovalbumin. J. Sci. Food

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high-intensity ultrasound on the structural and functional properties of α-Lactalbumin,

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β-Lactoglobulin and their mixtures. Food Res. Int. 2012, 48(2), 940-943.

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Ren, F., Glycation of α-lactalbumin with different size saccharides: Effect on protein

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structure and antigenicity. Int. Dairy J. 2014, 34(2), 220-228.

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protein glycation and antioxidant capacity of rare sugar− protein/peptide conjugates. J.

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Pang, J.; Yang, P., The reduction in the IgE-binding ability of β-Lactoglobulin by

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dynamic high-pressure microfluidization coupled with glycation treatment revealed

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by high-resolution mass spectrometry. J. Agric. Food Chem. 2017, 65(30),

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6179-6187.

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antioxidative activity of glycated α-lactalbumin with a rare sugar, D-allose, by

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Reduced immunogenicity of β-lactoglobulin by conjugation with carboxymethyl

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T., Glycation and phosphorylation of α-lactalbumin by dry heating: Effect on protein

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Alpha-lactalbumin: its production technologies and bioactive peptides. Compr. Rev.



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Identification of active peptides by HPLC-MS/MS. J. Agric. Food Chem. 2005, 53(3),

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523

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(40) Harsha, P. S.; Lavelli, V.; Scarafoni, A., Protective ability of phenolics from

529

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531


Page 24 of 36

Page 25 of 36


Figure captions Fig. 1: Size exclusion chromatography (SEC) for analyses N-LA, N-LA-Glc, LAGlc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150. Fig. 2: The intrinsic fluorescence spectra of N-LA, N-LA-Glc, LA-Glc-60, LA-Glc90, LA-Glc-120 and LA-Glc-150. Fig. 3: The ABTS radical-scavenging activity (%) of N-LA, N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150. Different letters on the top of the bars denote significant difference (p < 0.05). Fig. 4: Mass spectra for the unglycated peptides of LA-Glc-150. (A) peptide 61-71 at m/z 434.51073+, (B) peptide 91-102 at m/z 462.92253+, (C) peptide 104-115 at m/z +

+

485.93093 , (D) peptide 105-119 at m/z 604.65383 , (E) peptide 111-123 at m/z 570.29263+. The determined peptides are labelled by residue numbers. The m/z differences between glycated and unglycated peptides are indicated above the arrows. Fig. 5: The ETD MS/MS spectra of the glycated peptides. (A) the glycated peptide C(carbamidomethyl)KDDQNPHSSN71 with m/z of 488.52813+, (B)

61

C(carbamidomethyl)VKKILDKVGIN102 with m/z of 570.97853+, (C) the glycated

91

peptide 91C(carbamidomethyl)VKKILDKVGINY103 with m/z of 428.73784+, (D) the glycated peptide 104WLAHKALC(carbamidomethyl)SEKL115 with m/z of 539.94843+, (E) the glycated peptide 105LAHKALC(carbamidomethyl)SEKLDQWL119 with m/z of 658.67163+. (F) the glycated peptide 111

C(carbamidomethyl)SEKLDQWLC(carbamidomethyl)EKL123 with m/z of

624.29033+. The sequence of per peptide is depicted on the top of the spectrum. The



identified glycated sites are indicated by a line with glucose. The c and z ions are shown by the numbers and lines. Fig. 6: Ribbon diagram of the glycated α-LA (PDB 1F6S). The glycation sites are colored as follows: grey, framework of α-LA; red, glycation sites of the native α-LA; green, additional glycation sites of the α-LA after ultrasonication. Fig. 7: The average degree of substitution per peptide molecule (DSP) value of glycated sites of N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150.


Page 26 of 36

Page 27 of 36


Fig. 1: Size exclusion chromatography (SEC) for analyses N-LA, N-LA-Glc, LAGlc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150.

N-LA N-LA-Glc LA-Glc-60 LA-Glc-90 LA-Glc-120 LA-Glc-150

100

Relative absorbance

80

60

40

20

0 15

16

17

18

19

20

21

22

Elution time (min)


23

24

25


Page 28 of 36

Fig. 2: The intrinsic fluorescence spectra of N-LA, N-LA-Glc, LA-Glc-60, LA-Glc90, LA-Glc-120 and LA-Glc-150.

N-LA N-LA-Glc LA-Glc-60 LA-Glc-90 LA-Glc-120 LA-Glc-150

600

Fluorescence intensity

500

400

300

200

100

0 300

320

340

360

Wavelength (nm)


380

400

Page 29 of 36


Fig. 3: The ABTS radical-scavenging activity (%) of N-LA, N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150. Different letters on the top of the bars denote significant difference (p < 0.05).

0.7

e

d 0.6

c

bc

b

0.5 0.4

a

0.3 0.2 0.1


-1 50 lc G

LA -

LA -

G

lc

-1 20

-9 0

-6 0 lc

lc LA -G

-L N

LA -G

AG lc

-L A

0.0

N

ABTS radical-scavenging activity (%)

0.8


Page 30 of 36

Fig. 4: Mass spectra for the unglycated peptides of LA-Glc-150. (A) peptide 61-71 at m/z 434.51073+, (B) peptide 91-102 at m/z 462.92253+, (C) peptide 104-115 at m/z 485.93093+, (D) peptide 105-119 at m/z 604.65383+, (E) peptide 111-123 at m/z 570.29263+. The determined peptides are labelled by residue numbers. The m/z differences between glycated and unglycated peptides are indicated above the arrows.

AA (61-71)

A

100

80

Relative abundance

80

60

+3 434.5107

40

+3 m/z=54.0174 488.5281

20

80

60

40

AA (104-115)

C

+3 462.9225 m/z=54.0381

+3 570.9785

+3 516.9606

20

60

+3 485.9309

40

20

m/z=54.0175

+3 539.9484

m/z=54.0179 410

420

430

440

450

460

470

480

490

500

0 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600

mass (m/z)

100

AA (105-119)

D

100

80

460

470

480

490

+3 604.6538

40

20

+3 658.6716

AA (111-123)

E

600

610

620

+3 570.2926

20

m/z=54.0178

590

60

40

630

640

650

660

670

680

0 550

500

510

mass (m/z)

80

60

0 580

0 450

mass (m/z)

Relative abundance

0 400

Relative abundance

Relative abundance

100

AA (91-102)

B

Relative abundance

100

+3 624.2903 m/z=53.9977

560

570

580

mass (m/z)


590

600

610

mass (m/z)

620

630

640

650

520

530

540

550

Page 31 of 36


Fig.5: The ETD MS/MS spectra of the glycated peptides. (A) the peptide C(carbamidomethyl)KDDQNPHSSN71 with m/z of 488.52813+, (B) the peptide

61

C(carbamidomethyl)VKKILDKVGIN102 with m/z of 570.97853+, (C) the peptide

91

91

C(carbamidomethyl)VKKILDKVGINY103 with m/z of 428.73784+, (D) the peptide WLAHKALC(carbamidomethyl)SEKL115 with m/z of 539.94843+, (E) the peptide

104

105

+

LAHKALC(carbamidomethyl)SEKLDQWL119 with m/z of 658.67163 . (F) the

peptide 111C(carbamidomethyl)SEKLDQWLC(carbamidomethyl)EKL123 with m/z of 624.29033+. The sequence of per peptide is depicted on the top of the spectrum. The identified glycated sites are indicated by a line with glucose. The c and z ions are shown by the numbers and lines.

AA (61-71)

100

2 3 4 5 6 7 8 9 10 11

C KD D QN P H SS N 11 9 8 7 6 5 4 3 2

2 3 4 5 6 7 8 9 10 11 12

C VK K I L D KV G I N 12 1110 8 7 6

Glc

43 2

z

Glc

1326.7092,c81307.7015,z9 1425.7896,c9 1435.8795,z10 1482.8268,c10 1535.8318,z11+1 1595.8988,c11

6

1036.6016,c7 1017.6049,z8

40

20

0

676.3699,z5 695.5075,c4 791.4779,z6 809.5352,c5+1 921.6217,c 904.4004,z7

60

405.3303,c3

Relative abundance

1174.4836,c8

AA (91-102)

c

80

1261.4570,c9 1288.5302,z10+1 1348.6303,c10

997.2970,z9

882.2476,z8

B

z

Glc

1037.5393,c7

20

699.3088,c4+1 767.3072,z7

40

428.0748,z4 468.3496,c2

60

177.9824,c1

Relative abundance

80

826.2981,c5

c

277.2514,c2

A 583.2476,c3 639.2216,z6

100

0 200

400

600

800

1000

1200

1400

1600

200

400

mass (m/z)


600

800

1000

1200

mass (m/z)

1400

1600

1800

Relative abundance 40

20

200 400

80

60

600 800 1600

15 14 13 12 1110 9 8 7

Relative abundance

1433.9200,c11

1180.6198,z91164.6140,c8 1263.7394,c9 1309.6865,z10+1 1320.7831,c10

20

L A H K A L CS E K L D QW L

5 4 3 2

0 1000 1200 1400 1600 1800 2000 z

1800 200

100

20

200 400

mass (m/z)

ACS Paragon Plus Environment 400

80

Glc

80

600

mass (m/z) 600 800

F

60

40

800

c

12 11 10 9

1000

2 3 4 5 6 7 8 9 10 1112 13 14 15

mass (m/z)

1200

1504.7905，c11+1

1094.6017,z8 1159.6459,c8 1232.6746.z9+1 1246.6782,c9 1303.7139.z10+1 1375.7249,c10 1416.7567,z11+1

999.6705,c7

733.5787,z6 804.5662,z7 815.5378,c 5 886.5884,c6

622.5347,z5+1

460.5051,z4 525.5196,c4

373.4115,z3

244.2994,z2

z

1756.7581,c12

c 1400

40

c

1466.6117,c11 1479.6781,z10+1 1608.7542,z11+1

0

D

1350.6132,z9

1200

60

1122.5525,z7

717.5822,z112+ 774.4967,c122+

Glc

994.6263,z6 1064.4672,c8

1000

5 43 2

750.3962,c6

800 1067.5609,z8

759.5551,c6 838.4581,z +1 874.6055,c7 6 954.4203,z7

405.1248,c3

C VK K I L D KV G I N Y

808.3806,z5 878.6089,c7

E

13 12 11 10 9 8 7

100

406.3001,z2

600

AA (91-103)

522.3035,c4 535.3342,z 635.4223,c5 3

400

2 3 4 5 6 7 8 9 10 11 12 13

Relative abundance

200

c

1437.5723,z10 1509.6375,z11+1 1545.7932,c12 1636.6967,z12 1675.8352,c13+1 1774.1211,z13+1 1845.8638,z14+1

20

811.5647,c7 898.5483,c8 930.7515,c14+12+ 1028.6371,c9 1077.5889,z7 1164.6593,z8 1316.6409,c10+1

60 277.1412,c2

80

650.7637,c6+1

100

C

467.2661,c4

40

393.2061,z3 450.2657,z4 533.4204,c4 549.2681,z5 645.6258,c5+1

100

339.1884,c3

Relative abundance

Journal of Agricultural and Food Chemistry Page 32 of 36

2 3 4 5 6 7 8 9 10 11 12

AA (104-115)

W L A H K A L C S EK L Glc

7 6 54 3 2

1400

13 12 1110 9 8 7 6 5 4 3

C S E K L D QW LC E K L

z

0

mass (m/z)

1600

AA (105-119)

2 3 4 5 6 7 8 9 10 1112 13

AA (111-123)

Glc

z

0 1000 1200 1400 1600 1800 2000

Page 33 of 36


Fig. 6: Ribbon diagram of the glycated α-LA (PDB 1F6S). The glycation sites are colored as follows: grey, framework of α-LA; red, glycation sites of the native α-LA; green, additional glycation sites of the α-LA after ultrasonication.



Fig. 7: The average degree of substitution per peptide molecule (DSP) value of glycated sites of N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150.

N-LA-Glc LA-Glc-60 LA-Glc-90 LA-Glc-120 LA-Glc-150

1.0

0.8

DSP

0.6

0.4

0.2

0.0 61-71

91-102

104-115

105-119

peptide


111-123

Page 34 of 36

Page 35 of 36


Table 1. Summary of the glycated peptides in the N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150. Peptide

Sequence

488.52833+

1.09

(W)C*KDDQNPHSSN(I)

K62

91-102

570.9784

3+

0.70

(M)C*VKKILDKVGIN(Y)

K94,K98

91-103

428.73804+

0.53

(M)C*VKKILDKVGINY(W)

K98

61-71

488.52833+

0.07

(W)C*KDDQNPHSSN(I)

K62

91-102

570.9768

3+

2.69

(M)C*VKKILDKVGIN(Y)

K94,K98

91-103

428.73774+

-5.96


K98

104-115

539.94873+

1.79

(Y)WLAHKALC*SEKL(D)

K108

61-71

488.52803+

0.89

(W)C*KDDQNPHSSN(I)

K62

91-102

570.97823+

2.81

(M)C*VKKILDKVGIN(Y)

K94,K98

91-103

428.73764+

-6.31


K98

3+

-0.31

(Y)WLAHKALC*SEKL(D)

K108

61-71

488.52813+

0.55

(W)C*KDDQNPHSSN(I)

K62

91-102

570.97873+

3.04

(M)C*VKKILDKVGIN(Y)

K94,K98

91-103

571.3154

3+

1.34


K98

104-115

539.94873+

1.36

(Y)WLAHKALC*SEKL(D)

K108

105-119

494.25304+

-3.50

(W)LAHKALC*SEKLDQWL(C)

K114

61-71

488.52813+

0.96

(W)C*KDDQNPHSSN(I)

K62

91-102

570.97853+

1.11

(M)C*VKKILDKVGIN(Y)

K94,K98

91-103

428.73794+

-5.67


K98

104-115

539.9484

3+

-0.12

(Y)WLAHKALC*SEKL(D)

K108

105-119

658.67163+

0.66

(W)LAHKALC*SEKLDQWL(C)

K114

111-123

624.29043+

0.37

(L)C*SEKLDQWLC*EKL

K122

location

(m/z)

N-LA-G

61-71

LA-Glc-90

104-115 LA-Glc-120

LA-Glc-150

Glycated Δppm

Sample

LA-Glc-60

a

Glycated peptide

539.9499

C* refers to carbamidomethyl.


a

site


Page 36 of 36

Graphical abstract

Native α-LA Ultrasonic pretreatment Dry heating glycation with glucose

Dry heating glycation with glucose Glucose

Glycation extent

Antioxidant activity


Improved Antioxidant Activity and Glycation of Î±-Lactalbumin after

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