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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

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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


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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

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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

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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,

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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

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(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

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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

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

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

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glycation; FTICR-MS

2


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ABBREVIATIONS USED

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

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

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

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

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

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mass spectroscopy; ECD, electron capture dissociation.

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Introduction

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

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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

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

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

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

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

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

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chemical treatments, such as hydrolysis11,

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

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

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

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

12

and glycation;13 and genetic modification.14,

15

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

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

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

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

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such as antioxidant activity and allergenicity.18,

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

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19

However, a single modification could not

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


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

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

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

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

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

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

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

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

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glycation after ultrasound pretreatment remains ambiguous.

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

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

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

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

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

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

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

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

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

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

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

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method for β-Lg hyposensitization.

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Materials and methods

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Materials

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

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anti-human

IgE-HRP conjugate

(A9667),

1-Anilinonaphthalene-8-sulfonate (ANS)

and

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

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

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

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

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

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water. All chemicals used were of analytical grade.

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β-Lg samples preparation

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

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

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

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

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

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

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

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

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


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

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

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

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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

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

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

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

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Determination of IgG and IgE binding abilities

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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Far-UV CD spectroscopy

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

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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

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

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

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

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secondary structures were analyzed with DichroWeb.

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UV absorption and intrinsic fluorescence spectroscopy

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

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

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

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

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

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

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Surface hydrophobicity

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According to the method of Matulis and Lovrien

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with modification, the surface

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


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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

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

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

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

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fluorescence intensity to the protein concentration with different concentrations.

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HPLC-FTICR-MS analysis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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DSP =

∑ i×I ∑ I i −0 n

( peptide +i ×mannose )

i = 0 ( peptide+ i ×mannose )

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

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attached to the peptide in each glycated form.

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Statistical analysis

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

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

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Northampton, MA).

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Results and discussion

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IgG and IgE binding capacities analysis

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

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

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

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

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

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

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


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

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

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

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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

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

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

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

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

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the IgG and/or IgE binding of β-Lg.

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

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

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

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

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

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

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

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

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

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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

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

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



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

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

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

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Secondary structure analysis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


27

who showed that OVA

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

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

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

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ultrasound pretreatment combined with dry-state glycation.

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UV absorption and intrinsic fluorescence spectra analysis

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

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

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

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

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

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

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

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

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

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

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markedly loosened.18

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

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

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

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

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



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

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

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

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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

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

251

fluorescence spectra of β-Lg.

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Surface hydrophobicity

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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

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

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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

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

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

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

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

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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


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pretreatment.

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Glycation site determination

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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

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

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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.

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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

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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



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


Page 16 of 40

Page 17 of 40


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



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


Page 18 of 40

Page 19 of 40


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



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


Page 20 of 40

Page 21 of 40


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

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399

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Technol. 2011, 233, 387-394.

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

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

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

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

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

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

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

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

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

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

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

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

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

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Food Chem. 2011, 59, 5729-5737.

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

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

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

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

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

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

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

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

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Food Agric. 2015, 95, 2916-2923.

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

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

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

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

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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.

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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


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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



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


Page 29 of 40


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


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



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


Page 31 of 40


A


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


B

Fluorescence intensity

800 700 600 500 400 300 200 100 300

530

320

340

360

380

Wavelength (nm)

31


400

420


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


Page 33 of 40


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


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


FT[M+H] =583.9910 m/z

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

1600

1800


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


Page 35 of 40



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



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


Page 36 of 40

Page 37 of 40


A

B

AA (41-60)

AA (84-101) 4+

3+

50

0


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



3+

771.7538

0

760

780

800

820

4+

4+

50

604.5623

523.5362

520

540

560

580

840



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




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



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

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