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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
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Mechanism of the reduction in the IgG and IgE binding of β-lactoglobulin
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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,
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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: tuzc_mail@aliyun.com (Zongcai Tu); wanghui00072@aliyun.com (Hui Wang).
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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
17
0-600 W significantly reduced the IgG and IgE binding of β-Lg, with the lowest values observed
18
at 400 W. The decrease in the IgG and IgE binding of β-Lg was attributed to the increase in
19
glycation extent and the changes of secondary and tertiary structure, which reflected in the
20
increase of UV absorbance, α-helix and β-sheet contents as well as the decrease of intrinsic
21
fluorescence intensity, surface hydrophobicity, β-turn and random coil contents. Moreover,
22
ultrasound pretreatment promoted the reduction of IgG and IgE binding abilities by improving
23
glycation, reflecting in the increase of the glycation sites and the degree of substitution per peptide
24
(DSP) value determined by Fourier transform ion cyclotron resonance mass spectrometry
25
(FTICR-MS). Ultrasound pretreatment at 400 W showed the most significantly enhanced
26
glycation extent. Besides, the results suggested FTICR-MS could provide insights into the
27
glycation at molecular level, which was conducive to the understanding of the mechanism of the
28
reduction in the IgG and IgE binding of β-Lg. Therefore, ultrasound pretreatment combined with
29
dry-state glycation may be a promising method for β-Lg hyposensitization.
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Keywords: β-lactoglobulin; IgG and IgE binding abilities; ultrasound pretreatment; dry-state
31
glycation; FTICR-MS
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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
41
that causes about 90% IgE-mediated cow's milk allergy (CMA).1, 2 It is a 162-amino-acid globular
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protein with a molecular mass of 18.36 kDa.3 The formation of two disulfide bonds
43
(Cys66-Cys160 and Cys106-Cys119) stabilizes a so-called-barrel (or calyx) structure that formed
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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
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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
75
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
139 140
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
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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
217
stable structure. Similar results were also reported that glycation increased the α-helix and β-sheet
218
contents and decreased β-turn and random coil contents.13 Glycation could make conformation of
219
protein to a more stable state at the expense of β-turn or unordered structure.28 It may result in the
220
increase of α-helix and β-sheet contents and the decrease of β-turn and random coil contents due
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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
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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
231
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)
235
residues exposed on the surface of β-Lg molecule. The results may be attributed to the destruction
236
of the interactions that maintain the rigid native protein tertiary structure by ultrasound
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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
239
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
242
fluorescence emission maximum (λmax). However, when β-Lg was glycated, the λmax intensity
243
decreased from 842 (β-Lg-N) to 513 (β-Lg-M), and the glycated β-Lg after ultrasound
244
pretreatment showed lower λmax intensity, with a lowest value λmax intensity of 386 13
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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
247
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
250
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
253
The surface hydrophobicity of β-Lg samples are shown in Figure 3C. β-Lg-H had no
254
significant difference in the surface hydrophobicity with β-Lg-N (p < 0.05). When β-Lg was
255
glycated with mannose at ultrasound pretreatment at 0-600 W, the surface hydrophobicity declined
256
from 1673 (β-Lg-N) to 1465 (β-Lg-M), 1352 (β-Lg-M-200) 1280 (β-Lg-M-400) and
257
1333(β-Lg-M-600), respectively. The result was in accordance with previous reports that glycation
258
significantly reduced the hydrophobicity of β-Lg.23, 30 It may be attributed to the masking of some
259
hydrophobic groups induced by amino acid residues modification through covalent attachment of
260
mannose. What's more, ANS could strongly bind cationic groups, such as lysine (Lys) and
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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
263
conjugation of Lys and/or Arg residues with mannose, make ANS bind less cationic groups and
264
finally lead to the further reduction of surface hydrophobicity. Accordingly, glycated β-Lg with
265
ultrasound pretreatment had lower surface hydrophobicity than that without ultrasound 14
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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
270
enzyme due to the specifically cleavage on the carbon side of amino acids Lys and Arg. It cannot
271
cleave the site when the Lys or Arg is modified by glycation. To reduce the formation of
272
aggregation, the glycation was performed at 55 °C for 4 h (pH 8.0) under dry-state. No apparent
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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.
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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
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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
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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
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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
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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
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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
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Research and Development Program of China (863 Program) (No. 2013AA102205) and National
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Natural Science Foundation of China (NSFC) (No. 31560458).
396
References
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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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26. Li, Z.; Luo, Y.; Feng, L., Effects of Maillard reaction conditions on the antigenicity of
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with improved thermostability and preserved structural packing. Biotechnol. Bioeng. 2004, 86, 78-87.
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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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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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36. Li, X.; Yuan, S.; He, S.; Gao, J.; Chen, H., Identification and characterization of the antigenic site
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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
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continuous allergenic epitope of bovine β-lactoglobulin recognized by human IgE binding. Clin. Exp.
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40. Sélo; Clément; Bernard; Chatel; Créminon; Peltre; Wal, Allergy to bovine β-lactoglobulin:
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specificity of human IgE to tryptic peptides. Clin. Exp. Allergy 1999, 29, 1055-1063.
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41. Sélo, I.; Négroni, L.; Créminon, C.; Yvon, M.; Peltre, G.; Wal, J. M., Allergy to bovine
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β–Lactoglobulin: Specificity of human IgE using cyanogen bromide–derived peptides. Int. Arch.
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Allergy Immunol. 1998, 117, 20-28.
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42. Ma, X. J.; Chen, H. B.; Gao, J. Y.; Hu, C. Q.; Li, X., Conformation affects the potential
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allergenicity of ovalbumin after heating and glycation. Food Addit. Contam. Part A Chem. Anal.
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Control Expo. Risk Assess. 2013, 30, 1684-1692.
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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).
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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.
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518 100
A
Inhibition %
80
60 1.80
7.64
11.17 13.75
1.94
18.11
40
β-Lg-N β-Lg-H β-Lg-M β-Lg-M-200 β-Lg-M-400 β-Lg-M-600
20
0 -0.5
0.0
0.5
1.0
1.5
2.0
Log(inhibitor concentration)
519 100
B
Inhibition %
80
60 2.40
7.89 11.86 14.82 20.07
2.67 40
β-Lg-N β-Lg-H β-Lg-M β-Lg-M-200 β-Lg-M-400 β-Lg-M-600
20
0 -0.5
0.0
0.5
1.0
1.5
2.0
Log(inhibitor concentration)
520 521
Figure 1. The changes of IgG (A) and IgE (B) binding abilities of β-Lg induced by ultrasound
522
pretreatment coupled with glycation. The IgG and IgE binding abilities of β-Lg samples were
523
determined by inhibition ELISA. Pooled rabbit anti-β-Lg-sera or human anti-β-Lg-sera (50 µL per
524
well) were incubated separately with 1, 5, 10, 10, 40, 48 µg mL-1 of corresponding OVA samples
525
as inhibitors.
29
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[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
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Journal of Agricultural and Food Chemistry
A
β-Lg-N β-Lg-H β-Lg-M β-Lg-M-200 β-Lg-M-400 β-Lg-M-600
0.7
Absorbance
0.6
0.5
0.4
0.3
0.2
0.1 240
260
280
300
320
340
Wavelength (nm)
529 900
β-Lg-N β-Lg-H β-Lg-M β-Lg-M-200 β-Lg-M-400 β-Lg-M-600
B
Fluorescence intensity
800 700 600 500 400 300 200 100 300
530
320
340
360
380
Wavelength (nm)
31
ACS Paragon Plus Environment
400
420
Journal of Agricultural and Food Chemistry
1900
Page 32 of 40
C
Surface hydrophobicity (H0)
1800 1700
a
a
1600
b
1500
c
1400
c d
1300 1200 1100
531
β-Lg-N
β-Lg-H
β-Lg-M
β-Lg-M-200 β-Lg-M-400 β-Lg-M-600
532
Figure 3. The changes in UV absorption (A), intrinsic fluorescence (B) spectra and surface
533
hydrophobicity (C) of β-Lg induced by ultrasound pretreatment combined with glycation. Letters
534
(a-d) in the bars mean significantly different (p < 0.05).
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Journal of Agricultural and Food Chemistry
3+ 583.9910
A
Relative Abundance %
100
AA (1-14)
80
60 3+ 529.9745 3+ 638.0082
40 530
531
∆m/z=54.0172
20 ∆m/z=54.0165
0 520
530
540
550
560
570
580
590
600
610
620
630
640
650
m/z
535 20
B
AA (1-14) 3+
L I VTQTMKGLD I QK z12 z11 z10 z9 z8 z7 z6 z5 z4 z3 z2
c4, 444.3180
0 200
400
600
800
1000
1200
m/z
536
33
ACS Paragon Plus Environment
1400
c13, 1620.8877
c12, 1492.8291
c9, 1151.6340 z9, 1179.6051
z8, 1078.5574 c8, 1094.6126
z7, 947.5170
c7, 804.4648
c5, 572.3766 z5, 600.3477 z6, 657.3692 c6, 673.4243 z+2 12, 704.8593
z4, 487.2637
5
c3, 343.2704 z3, 372.2367
10
c11, 1379.7450 z11, 1408.7114
Mannose
c10, 1264.7181 z10, 1307.6637
15
c2, 244.2020 z2, 259.1527
Relative Abundance %
FT[M+H] =583.9910 m/z
c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13
1600
1800
Journal of Agricultural and Food Chemistry
15
C
AA (1-14) c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13
FT[M+H]3+=638.0082 m/z
L I VTQTMKGLD I QK
0 200
400
800
1000
1200
1400
c12, 1654.8819
z12, 1507.7798 c11, 1541.7978
c10, 1426.7709 z11, 1408.7114
c8, 1256.6653 z10, 1307.6637 c9, 1313..6868
Mannose
z9, 1179.6051
z8, 1078.5574
Mannose
z7, 947.5170 c7, 966.5176
z+2 13, 1507.7798
c6, 835.4771
600
c5, 734.4294
z5, 600.3477 c4, 606.3708 z6, 657.3692
z4, 487.2637 c3, 505.3232
z3, 372.2367 c2, 406.2548
5
z2, 259.1527
10
c+2 13, 891.9620
z13 z12 z11 z10 z9 z8 z7 z6 z5 z4 z3 z2
c1, 293.1707
Relative Aboundance %
Page 34 of 40
1600
1800
m/z
537 538
Figure 4. Mass spectrum of the peptide 1-14 (A) and ECD MS/MS spectra of the glycated peptide
539
1-14 with m/z of 583.99103+ (B) and 638.00823+ (C) from the glycated β-Lg after ultrasound
540
pretreatment at 400 W. The m/z differences are indicated with numbers and arrows. The sequence
541
of peptide is shown on the top of figure. The determined glycation site (blue bold) is shown by an
542
arrow with mannose. The c and z ions are indicated by the number.
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Journal of Agricultural and Food Chemistry
Relative Abundance %
120
A
AA (41-60) 3+ 825.7706
100
80
60
40
3+ 771.7538
20
∆m/z=54.0168
0 750
760
770
780
790
800
810
830
840
850
m/z
543
20
B
AA (41-60) c10c11c12c13c14c15c16
c8
VYVEELKPTPEGDLEILLQK
3+ 15 FT[M+H] =825.7707 m/z
z15 z14
z10 z9 z8 z7 z6 z5 z4 z3 z2
z12
Mannose
0 200
400
600
800
1000
1200
1400
1600
1800
c16, 1991.9867
z15, 1840.0073 c15, 1878.9005
z14, 1726.9134 c14, 1749.8570
c13, 1636.7741
c12, 1521.7559
c11, 1464.7324
c10, 1335.7042 z12, 1339.7125
c8, 1173.5964 z10, 1141.6129
z8, 955.5525
z9, 1012.5746
z7, 840.5276
z6, 727.4442 c6, 750.3996
z5, 598.4027 c5 637.3167
z2, 259.1523
5
z4, 485.3191 c4, 508.2745
10
z3, 372.2356
Relative intensity %
c4 c5 c6
544
820
2000
m/z
545
Figure 5. Mass spectrum of the peptide 41-60 (A) and ECD MS/MS spectrum of the glycated
546
peptide 41-60 with m/z of 825.77073+ (B) from the glycated β-Lg after ultrasound pretreatment at
547
400 W. The m/z differences are indicated with numbers and arrows. The sequence of peptide is 35
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
548
shown on the top of figure. The determined glycation site (blue bold) is shown by an arrow with
549
mannose. The c and z ions are indicated by the number.
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Page 37 of 40
Journal of Agricultural and Food Chemistry
A
B
AA (41-60)
AA (84-101) 4+
3+
50
0
Relative Abundance %
771.7539
100
825.7706
760
780
800
820
840
3+
100
3+
771.7540
825.7707
50
0
760
780
800
820
4+ 4+
523.5363
604.5625
50
0
520
540
560
580
Relative Abundance %
Relative Abundance %
3+
771.7538
0
760
780
800
820
4+
4+
50
604.5623
523.5362
520
540
560
580
840
Relative Abundance %
Relative Abundance %
550
3+
50
780
800
820
640
50
4+
564.0493 4+
523.5364
0 500
520
540
560
580
600
620
640
564.0494
825.7704
760
620
4+
771.7537
0
600
604.5625
100
3+
100
640
4+
825.7706
50
620
4+
3+
100
600
564.0492
100
0 500
840
564.0494
100
500
Relative Abundance %
Relative Abundance %
Relative Abundance %
3+
840
100
4+
604.5624
50 4+
523.5362
0 500
520
540
m/z
560
580
600
620
640
m/z
551
Figure 6. Mass spectra of the glycated peptides 41-60 (A) and 84-101 (B). The red, blue, magenta
552
and green spectra refer the peptides from glycated β-Lg with ultrasound pretreatment at 0, 200,
553
400 and 600 W, respectively.
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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