Subscriber access provided by UNIV OF DURHAM
Functional Structure/Activity Relationships
Ultrasonic pretreatment combined with dry-state glycation reduced the IgE/IgG-binding ability of #lactalbumin revealed by high-resolution mass spectrometry Jun Liu, Zong-cai Tu, Guang-xian Liu, Chen-di Niu, Hong-lin Yao, Hui Wang, Xiao-mei Sha, Yan-hong Shao, and Igor A. Kaltashov J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00489 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 34
Journal of Agricultural and Food Chemistry
1
Ultrasonic pretreatment combined with dry-state glycation reduced
2
the IgE/IgG-binding ability of α-lactalbumin revealed by
3
high-resolution mass spectrometry
4 5
Jun Liua, Zong-cai Tua,b*, Guang-xian Liua, Chen-di Niuc, Hong-lin Yaoc, Hui Wangb*,
6
Xiao-mei Shaa, Yan-hong Shaoa, Igor A. Kaltashovc
7 8 9
a
College of Life Sciences, Jiangxi Normal University, Nanchang, Jiangxi 330022,
10
China;
11
b
12
Nanchang, Jiangxi 330047, China;
13
c
14
01003, USA
15
*
16
E-mail:
17
(Hui-Wang)
State Key Laboratory of Food Science and Technology, Nanchang University,
Department of Chemistry, University of Massachusetts-Amherst, Amherst, MA,
Corresponding authors. Tel.: +86-791-8812-1868; fax: +86-791-8830-5938.
[email protected] (Zong-cai
Tu),
18 19 20 21 22 23
ACS Paragon Plus Environment
[email protected] Journal of Agricultural and Food Chemistry
24
Abstract: Bovine α-lactalbumin (α-LA) is one of major food allergens in cow's
25
milk. The present work sought to research the effects of ultrasonic pretreatment
26
combined with dry heating-induced glycation between α-LA and galactose on the
27
IgE/IgG-binding ability and glycation extent of α-LA, which determined by inhibition
28
ELISA and high-resolution mass spectrometry respectively. The IgE/IgG-binding
29
ability of glycated α-LA were significantly decreased as a result of ultrasonic
30
pretreatment, while average molecular weight, incorporation ratio (IR) value, the
31
location and number of glycation site, and degree of substitution per peptide (DSP)
32
value was elevated. When the mixtures of α-LA and galactose pretreated by
33
ultrasonication at 150 W/cm2, the glycated α-LA possess seven glycation sites, the
34
highest IR and DSP value, and the lowest IgE/IgG-binding ability. Therefore, the
35
decrease in IgE/IgG-binding ability of α-LA depend not only on the shielding effect
36
of the linear epitope was found to be caused by the glycation of K13, K16, K58, K93
37
and K98 sites, but also on the intensified glycation extent, which reflected in the
38
increase of IR value, the number of glycation sites and DSP value. Moreover,
39
allergenic proteins and monosaccharides pretreated by ultrasonication then followed
40
by dry-state glycation was revealed as a promising way of achieving lower
41
allergenicity of proteins in food processing.
42 43
Keywords: Alpha-lactalbumin, IgE/IgG-binding ability, ultrasonication, glycation, mass
44
spectrometry
ACS Paragon Plus Environment
Page 2 of 34
Page 3 of 34
Journal of Agricultural and Food Chemistry
45
Introduction
46
Bovine α-lactalbumin (α-LA) is a potential allergen that causes about 30-35%
47
IgE-mediated cow's milk allergy1. Native α-LA is a 14.2 kDa Ca-binding protein,
48
which has 123 amino acid residues (AA) 2. It consists of a α-helical domain and a
49
β-sheet domain, which are connected by a calcium binding loop3. α-LA strongly binds
50
metal ions, possess a variety of such useful functional characteristics as
51
immune-modulating, antioxidant, antibacterial or antitumor activity4-6. The antigenic
52
site of α-LA was located on sequence 5-182, and a high IgE-binding ability is
53
associated with sequence (AA 17-58) of bovine α-LA7. Reported methods, such as
54
heat
55
non-enzymatic glycosylation (glycation)10, 11 rationally decreased the allergenicity of
56
α-LA. For these processes, glycation is the early stage of Maillard reaction, and
57
mainly occurs between a carbonyl group of saccharides and an amino group (Lys and
58
Arg) of proteins. It can improve the structural and physicochemical properties of
59
proteins11-14, for example, antioxidant ability, emulsifying, and foaming property,
60
especially for decreasing the allergenicity of α-LA14,
61
supplement for infant formulae which undergo the modification of glycation, the
62
functionality of α-LA was modulated in food processing. However, a single glycation
63
cannot reduce the allergenicity of α-LA to a satisfactory result.
treatment8,
gamma
irradiation1,
high-intensity
ultrasonication9
and
15
. α-LA can be used as a
64
Ultrasonication, a non-thermal processing technology, which can be used to
65
improve the glycation reaction16 and develop a new product with an unique
66
functionality17 in four characteristic ways18, including heating effects, acoustic
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 34
67
cavitation,
68
ultrasonication can disrupt the structure of α-LA19, 20, and glycation was strongly
69
decreased the IgE/IgG-binding ability of α-LA21, no study on the effects of ultrasonic
70
pretreatment coupled with glycation reaction on the IgE/IgG-binding ability of α-LA.
acoustic
streaming,
and
fluid
particles
oscillations.
Although
71
The shielding effects of the linear epitopes was caused by glycation reaction
72
between allergenic proteins and sugar, leading to influence the IgE/IgG-binding
73
ability of allergenic proteins. Glycation occurs on the Lys, Arg and N-terminal amino
74
acid of proteins and alters the peptides. The major α-LA epitopes probably contain
75
one or more Lys and Arg residues. Thus, the location of glycation site was associated
76
with the IgE/IgG-binding ability of α-LA. But, whether the change of the
77
IgE/IgG-binding ability of α-LA treated by ultrasonic pretreatment combined with
78
glycation are due to location and number of glycation sites is still uncertain. Moreover,
79
studies on the relationship between the IgE/IgG-binding ability and glycation extent
80
of α-LA that was ultrasonicated by ultrasonication and subjected to glycation are rare.
81
At present, high-resolution mass spectrometry such as Q Exactive mass spectrometer
82
can be used to investigate their relationships due to it can exactly analyse the number
83
and location of glycation site and glycation extent per site in the protein.
84
In the present study, ultrasonication was used to pretreat α-LA, aiming to study
85
the impact of ultrasonication on the IgE/IgG-binding ability, and on structural
86
properties of glycated α-LA. The first, the IgE/IgG-binding ability of glycated α-LA
87
was detected by inhibition ELISA. The glycation extent (illustrated by average
88
molecular weight, incorporation ratio value, the location and number of glycation
ACS Paragon Plus Environment
Page 5 of 34
Journal of Agricultural and Food Chemistry
89
sites, and degree of substitution per peptide value) of glycated α-LA were determined
90
using high-resolution mass spectrometry. Our study results understood that glycation
91
extent plays an essential role in reducing the IgE/IgG-binding ability of α-LA treated
92
by ultrasonic pretreatment coupled with glycation, thus provide basic information on
93
the potential applications of α-LA in the food industry and dairy products.
94
Materials and methods
95
Chemicals and materials
96
Alpha-lactalbumin (α-LA) from bovine milk (L5385, Type I, ≥ 85%), galactose
97
(G0625), pepsin (P6887, 3,200-4,500 units/mg protein), and Goat anti-human
98
IgE-HRP conjugate (A9667), mass Standards Kit for the 4700 Proteomics Analyzer
99
(AB SCIEX, 4333604) were from Sigma-Aldrich (St. Louis, MO). Goat anti-rabbit
100
IgG-HRP conjugate (SE131) was from Beijing Solarbio Technology Co., Ltd (Beijing,
101
China).
102
Ten sera from patients allergic to milk were from Plasma Lab International
103
(Everett, USA). They had total milk protein-specific IgE levels ranging from 5.74 to
104
78.6 KUA/L. Human antisera (prepared by mixed ten patient’s sera at same volume)
105
was applied to study the IgE-binding abilities of α-lactalbumin. Rabbit antisera was
106
prepared using a previously reported protocol1.
107
Sample Preparation
108
1.0 mg/mL of α-LA solution was prepared by dissolving native α-LA in 50 mM
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
109
phosphate buffer saline (PBS), and the pH of this solution was set to 7.4. Native α-LA
110
was used as the control. 10 mL of α-LA solution were transferred into 25 mL flat
111
bottom conical flasks which were immersed in the ice bath, followed by
112
ultrasonicated using a Q700 Sonicator (microtip probe (1/8 in. = 3 mm) with a 9s on
113
and 1s off pulsation at an actual ultrasonic intensity of 0, 90 and 150 W/cm2 for 15
114
min, respectively. Then, 10 mg of galactose (Gal) was prepared in 10 mL of native
115
and treated α-LA solution, respectively. 10 mg of galactose were dispersed in 10 mL
116
of native α-LA solution, and the subsequent procedure was the same to ultrasonic
117
treatment. Native α-LA solution, native α-LA -Gal solution, ultrasonicated α-LA -Gal
118
solution and ultrasonicated (α-LA -Gal) solution were freeze-dried into the powders,
119
followed by incubation at 55 oC and 79% relative humidity (saturated potassium
120
chloride solution) for 4 h. The reaction was stopped in an ice bath, then the samples
121
was filtered using a Centricon (Millipore) centrifugal filters with 3000 Da to remove
122
unreacted galactose and salts. The concentration of all the samples were diluted into
123
5.0 mg/mL for future use. Native α-LA was named N-LA. α-LA was treated by
124
ultrasonication at 0, 90 and 150 W/cm2 then glycation was named N-LA-G,
125
U-LA-G-90, U-LA-G-150, respectively. The mixtures of α-LA and Gal were treated
126
by ultrasonication at 90 and 150 W/cm2 then glycation was named U-(LA-G)-90 and
127
U-(LA-G)-150. The treatments were performed in triplicates. Schematic depiction of
128
the sample preparation was shown in Fig. 1.
129
IgE/IgG-binding ability determination
ACS Paragon Plus Environment
Page 6 of 34
Page 7 of 34
Journal of Agricultural and Food Chemistry
130
Inhibition ELISA assays were used to estimate the IgE/IgG-binding ability of α-LA
131
by the method of Chen et al.22, with human antisera and rabbit antisera, respectively.
132
The 96-wells microtitre plates were coated with 4 µg/mL of native α-LA samples
133
(100 µL/well) and followed by incubation at 4 oC for overnight. The plates were
134
washed three times by addition of PBST (prepared by dissolving 0.05% Tween-20 in
135
10 mM PBS) and followed by blocking by addition of 3% mg/mL fish gelatin
136
(dissolved in carbonate buffer) for 1 h at 37 oC, then again washed. The incubation at
137
37 oC for 2 h was initiated by addition of 50 µL of antisera samples (1:10 diluted
138
human sera or 1:10 000 diluted rabbit sera) and 50 µL of the treated samples. After
139
incubation, removed the solution, washed the plate. 100 µL of purified goat
140
anti-human IgE-HRP conjugate or goat anti-rabbit IgG-HRP conjugate (diluted into
141
1:5000 in PBST) were added, then incubated at 37 oC for 1 h. After incubation,
142
tetramethylenbenzidine solution (100 µL) were immediately added to each well, and
143
the reaction was stopped by addition of sulfuric acid (50 µL, 2 mol/L). Finally, the
144
absorption was monitored at 450 nm by a microplate reader (BioTek Instruments Co.
145
Ltd., Vermont). Decline rate was calculated: % inhibition = 1 − × 100 ,
146
where B and B0 are the absorbance value of the well with and without glycation
147
samples, separately. Each sample was performed in triplicates.
148
MALDI-TOF analysis
149
Molecular weight (MW) of all the glycated samples were analyzed using
150
MALDI-TOF mass spectrometer (AB Science, USA) according to a previously
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 34
151
reported method16. Matrix was prepared by dissolving 5.0 mg/mL of sinapic acid in
152
50% acetonitrile with 0.1% trifluoroacetic acid. The proteins were diluted into 1:100
153
with ultrapure water. 1.5 µL of mixtures at 1:1 protein solution to matrix ratio was
154
spotted onto MALDI target, then flowed by air-drying. Calibration Mixture 1 was set
155
as a standard calibration, error is less than 0.5 Da. The spectrum was acquired in
156
5000-50000 m/z range with 500 laser shots were accumulated for each measurement.
157
Data Explorer (TM) Software was applied to analyze the mass spectrometric data.
158 159
In this work we used incorporation ratio (IR) to analyze the degree of protein glycation. IR of Gal to α-LA can be estimated using the following equation:
160
IR =
MW
− MW"#$ 162.0528 !
!
161
where 162.0528 is molecular weight of Gal attached to α-LA, MWGlycated α-LA is the
162
MW of glycated α-LA, and MWUnglycated α-LA is the molecular weight of unglycated
163
α-LA.
164
Identification of glycation sites
165
The glycation sites of glycated α-LA were identified by our previous method23.
166
Protein digestion was prepared by filter-aided sample preparation method. After
167
filter-aided sample preparation, the peptides were separated with an Agilent 1200
168
HPLC (Agilent Technologies, USA) using a C18 column, then the column effluent
169
was performed by ETD-MS/MS for samples analysis, as previously reported setup
170
and method23.
ACS Paragon Plus Environment
Page 9 of 34
Journal of Agricultural and Food Chemistry
171 172
We applied the degree of substitution per peptide of each site (DSP) to analyze glycation extent. DSP can be estimated using the following equation24: ∑#/34 i × I../0 / × 12 DSP = ∑#/34 I../0 / × 12
173
where I is the sum of the intensity of glycated peptide, and i is the number of
174
galactose units attached to the peptide in each glycated form.
175
Results and Discussion
176
Analysis of IgE/IgG-binding ability
177
The IgE/IgG-binding ability of N-LA, N-LA-G, U-(LA-G)-90, U-(LA-G)-150,
178
U-LA-G-90 and U-LA-G-150 were determined with inhibition ELISA assays. The
179
IgE/IgG-binding ability was reflected by IC50 value. The results are listed in Fig. 2.
180
The IC50 value of U-(LA-G)-90, U-(LA-G)-150, U-LA-G-90 and U-LA-G-150 shifted
181
to 14.5, 16.2, 12.2, and 14.2 µg/mL, respectively, much higher than the IgE-binding
182
ability of N-LA and N-LA-G, which were 4.5 and 11.1 µg/mL (Fig. 2A). Interestingly,
183
Fig. 2B shows a similar trend. The IC50 value of N-LA-G (3.9 µg/mL), U-(LA-G)-90
184
(5.5 µg/mL), U-(LA-G)-150 (6.4 µg/mL), U-LA-G-90 (4.6 µg/mL) and U-LA-G-150
185
(5.1 µg/mL) were higher than that of N-LA (1.1 µg/mL). These results indicated that
186
α-LA treated by different ultrasonic powers combined with glycation gave a higher
187
IC50 value than native α-LA and untreated samples. It implies glycation can reduce the
188
IgG/IgE-binding abilities of α-LA, and ultrasonic pretreatment promoted the
189
reduction. The decrease in IgG/IgE-binding abilities of α-LA due to partial shielding
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
190
of some linear epitopes by conjugation with reducing galactose21, 25, and the structural
191
changes induced by glycation. Previous work reported that secondary and tertiary
192
structure of α-LA was disrupted by ultrasonication20, and ultrasonication can increase
193
glycation site(s) of α-LA and BSA16, 23. Yang et al. reported that ultrasonic treatment
194
combined with glycation can reduce the IgE/IgG-binding abilities of β-Lactoglobulin
195
by increase in glycation extent26. As show in the Fig. 2, the decreased
196
IgE/IgG-binding ability of α-LA under ultrasonication combined with glycation
197
treatment was also observed. Therefore, ultrasonic treatment combined with glycation
198
can also reduce the IgE/IgG-binding ability of α-LA by the increase in the glycation
199
extent and disrupt the structure. In order to understand their relationship clearly, the
200
glycation extent (illustrated by IR value, the number and location of glycation site,
201
and DSP value) were investigated using mass spectrometry in the subsequent
202
experiments.
203
Incorporation ratio (IR) of Gal to α-LA
204
Fig. 3 presents mass spectrometry analysis of all glycated samples. Average
205
molecular weights of N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LA-G-90 and
206
U-LA-G-150 shifted to 14987.07, 15284.94, 15309.74, 15153.45 and 15168.48 Da,
207
respectively, much higher than the mass of the polypeptide backbone of α-LA,
208
14186.06 Da. Their IR was respectively calculated as 4.94, 6.78, 6.92, 5.97, 6.06.
209
This finding demonstrated that ultrasonic treatment significantly improved the
210
glycation degree of α-LA. However, MALDI TOF-MS can only measure intact
ACS Paragon Plus Environment
Page 10 of 34
Page 11 of 34
Journal of Agricultural and Food Chemistry
211
molecular weight of the treated α-LA, the location and number of glycation sites and
212
DSP value could be not revealed. To fully investigate them, HPLC-ETD MS/MS was
213
performed after pepsin digestion.
214
The location and number of glycation sites determination
215
If a peptide was glycated by galactose, and corresponding m/z of peak with the
216
charges of 1, 2, 3, 4, or 5 will appear mass shift 162.0528, 81.0264, 54.0176, 40.5132,
217
or 32.4106 accordingly. The results of mass spectrum analysis are shown in Fig. 4.
218
The m/z peaks of non-glycated peptide 9-23, 10-23, 53-71, 92-103, and 104-117 were
219
561.31393+, 512.29063+, 587.02334+, 463.94373+, 566.95903+, whereas the relative m/z
220
peaks of glycated peptide were 615.33143+, 566.30823+, 668.04974+, 571.98863+,
221
620.97673+, separately. The m/z shift of these peaks were 54.0175, 54.0176, 81.0264,
222
108.0449, and 54.0177 Da separately. This result indicated that these peptides had
223
mono-glycated, or dual-glycated peptides.
224
As we known, there are 12 Lys, 1 Arg and N-terminal of Asn45 in the native
225
α-LA, whence α-LA includes 13 potential glycation sites. We used HPLC-ETD
226
MS/MS to obtain the detailed map of glycation site K13, K16, K58, K62, K93, K98,
227
and K108 of α-LA (Fig. 5). Fig. 5A presents ETD MS/MS spectrum of
228
mono-glycated peptide 9FRELKDLKGYGGVSL23 with a peak at m/z of 615.33143+.
229
K13 was obtained by the mass difference between the c4 and c6 ions, or between the
230
z10 and z12, confirming that Gal was attached to K13. K16, K98 and K108 were also
231
determined by mass spectrometry of glycated peptide with m/z of 566.30823+,
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 12 of 34
232
428.73764+, 620.97673+, respectively. Fig. 5B, 5D and 5F presents the c and z ions
233
were
234
91
235
104
236
5E
237
53
238
with m/z of 668.04974+, 571.98863+ were identified to be K58, K62, K93 and K98
239
respectively. These results indicated that HPLC-ETD MS/MS can identify the
240
location of glycation sites of α-LA clearly.
highly
matched
with
the
10
peptides
RELKDLKGYGGVSL23,
C(carbamidomethyl)VKKILDKVGINY103
and
WLAHKALC(carbamidomethyl)SEKLDQ117, respectively. Similarly, Fig. 5C and presents
ETD
MS/MS
of
the
FQINNKIWC(carbamidomethyl)KDDQNPHSSN71
dual-glycated and
92
peptides
VKKILDKVGINY103
241
Glycated peptide and glycation sites of N-LA-G, U-(LA-G)-90, U-(LA-G)-150,
242
U-LA-G-90 and U-LA-G-150 was summarized in Table 1. N-LA-G contains five
243
glycation sites, including K13, K16, K93, K98 and K108. We used ultrasonication at
244
90 and 150 W/cm2 to pretreat α-LA, six sites (K13, K16, K62, K93, K98, and K108)
245
were glycated. However, the mixtures of α-LA and Gal were treated at same
246
ultrasonic conditions, two additional glycated sites (K58 and K62) were found to be
247
glycated. The results were consistent with average molecular weight and IR value of
248
the treated α-LA (Fig. 3). The increase of glycation sites was probably because
249
structure of α-LA loosened with ultrasonic treatment, which accelerated glycation,
250
and exposed more reactive sites16, 27, 28. In present study, K58 was not observed in
251
U-LA-G-90 and U-LA-G-150, but it was detected in the U-(LA-G)-90 and
252
U-(LA-G)-150. Intriguingly, no obvious difference existed between the glycation site
253
of U-LA-G-90 and U-LA-G-150, the same result was found in the U-(LA-G)-90 and
ACS Paragon Plus Environment
Page 13 of 34
Journal of Agricultural and Food Chemistry
254
U-(LA-G)-150, suggesting that ultrasonic pretreatment can change the structure of
255
α-LA, confirmation of Gal and promote the glycation reaction between α-LA and Gal
256
as well. These results indicated that glycation sites were associated with the
257
pre-treated α-LA and galactose by ultrasonication. Fig. 6 shows that two additional
258
sites (K58 and K62) were found in the treated α-LA after ultrasonic treatment
259
combined with glycation. A very similar result was exhibited by the previous study,
260
where both glycated α-LA and BSA mainly occurred on Lysine29, 16. Therefore, it
261
seems credible that the number of glycation site can be affected by conformational
262
changes of α-lactalbumin treated by ultrasonic treatment.
263
Ultrasonication on the DSP value of α-LA
264
Fig. 7 shows the DSP value for all glycated peptides of N-LA-G, U-(LA-G)-90,
265
U-(LA-G)-150, U-LA-G-90 and U-LA-G-150. After ultrasonic treatment, glycated
266
peptide exhibited a higher DSP than that of un-treated samples. For example, K13 is
267
the reactive Gal glycation sites in N-LA-G with DSP value close to 0.19, however,
268
ultrasonic treatment increased their DSP value to 0.37, 0.64, 0.34 and 0.5 in the
269
U-(LA-G)-90, U-(LA-G)-150, U LA-G-90 and U-LA-G-150 respectively. Similarly,
270
the DSP value of other glycated peptides were increased by ultrasonic treatment. This
271
is because the structural change of α-LA by ultrasonic treatment, thus, gaining more
272
accessibility to the glycation, as had been previously demonstrated16. Interestingly, we
273
found the peptides of U-(LA-G)-90 had a higher DSP value compared to that of
274
U-LA-G-90, the similar phenomenon was observed between U-(LA-G)-150 and
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
275
U-LA-G-150. It was likely that α-LA and Gal were treated by ultrasonication, maybe
276
changed their conformation, and finally caused the increase in the glycation between
277
α-LA and Gal.
278
Analysis of ultrasonic pretreatment combined with glycation reduced
279
the IgE/IgG-binding abilities of α-LA
280
It is important to note that extent of Maillard reaction play an essential role in
281
explaining functional behaviors of Maillard reaction products (MRPs). In our recent
282
study, combining ultrasonic pretreatment with glycation observably reduced the
283
IgG/IgE-binding ability of α-LA, which was closely related to shielding effect of the
284
linear epitopes, the location of glycation sites, IR and DSP value. To explore this
285
result, high-resolution mass spectrometry can be employed to study the modification
286
of acid amino sequence, the location of glycation sites, and DSP value.
287
In the glycation reaction, saccharides can affect protein allergy, mainly by
288
masking the linear epitopes25. Järvinen et al.30 found that potential α-LA allergenic
289
epitopes identified are the fragments of the peptide 1-16, 13-26, 47-58, and 93-102.
290
These sequences contain one or more lysine residues (K13, K16, K58, K93, K94 and
291
K98), the glycation reaction between Lys and Gal results in modification of linear
292
epitopes with significant reduction of IgE/IgG reactivity. In this study, without
293
ultrasonic treatment, four sites (K13, K16, K93 and K98) were glycated in the
294
glycation between native α-LA and Gal, and results in linear epitopes changes, finally
295
led to a higher IC50 value compared to un-glycated samples (Fig. 2). As shown in
ACS Paragon Plus Environment
Page 14 of 34
Page 15 of 34
Journal of Agricultural and Food Chemistry
296
Table 1 and Fig. 6, K13, K16, K93 and K98 were discovered in all the glycated
297
samples, indicating that these epitopes were necessary to reduce the IgE/IgG-binding
298
ability of α-LA. It can be explained by the glycation of K13, K16, K93 and K98 sites
299
can mask the epitopic areas of α-LA. We applied ultrasonication at 90 and 150 W/cm2
300
to pretreat α-LA, one additional glycated site (K62) of glycated α-LA was found (Fig.
301
6). Although the epitopic areas of α-LA does not include K62, it can also alter acid
302
amino sequence of α-LA, and may be related to the IgE/IgG-binding ability. When the
303
mixtures of α-LA and Gal were ultrasonicated under same conditions, two additional
304
glycated sites K58 and K62 were identified (Fig. 6), which led to the conformation of
305
the linear epitopes changes, enhancing the IC50 value (Fig. 2), finally decreasing the
306
IgE/IgG-binding ability of α-LA. This suggests that high intensity ultrasonication uses
307
can bring about conformational changes of α-LA and Gal31, 32, improved the glycation
308
reaction and glycation sites, thereby decreased the IgE/IgG-binding ability. By
309
comparing IC50 value of all samples, glycation site K58 was identified in the
310
U-(LA-G)-90 and U-(LA-G)-150, which the IgE/IgG-binding ability reductions were
311
higher than that of U-LA-G-90 and U-LA-G-150. A recent study by Zhang et al.21
312
reported that glycation of K58 was dominant in α-LA-glucose, which the decreased
313
antigenicity was the highest. These findings indicated that the location and number of
314
glycation sites can significantly affect the IgE/IgG-binding ability of α-LA.
315
Although U-(LA-G)-90 and U-(LA-G)-150 had same number of glycation sites
316
(Table 1), U-(LA-G)-150 had a higher IR, DSP and IC50 value compared to those of
317
U-(LA)-G-90. The similar result was observed between U-LA-G-150 and U-LA-G-90
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
318
(Fig. 2, Fig. 3 and Fig. 7). Thus, α-LA underwent a reaction regions exposure after
319
ultrasonication treatment, these reaction regions were well exposed with increased
320
ultrasonic power, which improved the glycation extent of α-LA, eventually reduced
321
its potential allergenicity. These results indicated that the IgE/IgG-binding abilities of
322
glycated α-lactalbumin could be affected by the IR and DSP values. Additionally, the
323
structure of α-LA was changed by heat treatment, leading to the disruption of linear
324
epitopes33, finally impacting the IgE/IgG-binding ability of α-LA. Therefore,
325
ultrasonic treatment coupled with glycation decreased IgE/IgG-binding ability of
326
α-LA by both masking the epitopes, and by improving its glycation extent.
327
Conclusions
328
In this paper, combining ultrasonic pretreatment with glycation caused much
329
greater reduction in IgE/IgG-binding abilities of alpha-lactalbumin compared to the
330
individual dry-sate glycation alone. The result was attributed to the glycation of K13,
331
K16, K58, K93 and K98 sites can mask the linear epitopes of α-LA. Furthermore,
332
ultrasonic treatment promoted the reduction in IgE/IgG-binding ability of α-LA by
333
increasing the amount of glycation sites, IR and DSP value. However, some
334
experiments in vivo and vitro, such as double-blind placebo-controlled trial, and cells
335
experiment, need be measured to fully ensure the reduced allergenicity of glycated
336
α-LA during food processing.
337
Acknowledgements
ACS Paragon Plus Environment
Page 16 of 34
Page 17 of 34
Journal of Agricultural and Food Chemistry
338
This work was supported by the earmarked fund for China Agriculture Research
339
System
340
(20162BCB23017) and Chinese National Natural Science Foundation (No. 31460395
341
and 31760440).
342
Abbreviations
343
α-LA, alpha-lactalbumin; IgE, immunoglobulin E; IgG, immunoglobulin G; Gal,
344
Galactose; DSP, the degree of substitution per peptide; ELISA, enzyme-linked
345
immunosorbent assay; AA, amino acid; MALDI TOF, matrix-assisted laser desorption
346
ionization time of flight; HPLC, high performance liquid chromatography; ETD
347
MS/MS, electron transfer dissociation mass spectrometry/mass spectrometry.
348
References
(CARS-45),
Excellent
Youth
Foundation
of
Jiangxi
Province
349 350
[1] Meng, X.; Li, X.; Wang, X.; Gao, J.; Yang, H.; Chen, H., Potential allergenicity
351
response to structural modification of irradiated bovine α-lactalbumin. Food Funct.
352
2016, 7(7), 3102-3110.
353
[2] Wal, J. M., Cow's milk allergens. Allergy 1998, 53(11), 1013-1022.
354
[3] Permyakov, E. A.; Berliner, L. J., α‐Lactalbumin: structure and function. FEBS
355
Lett. 2000, 473(3), 269-274.
356
[4] Sedaghati, M.; Ezzatpanah, H.; Mashhadi Akbar Boojar, M.; Tajabadi Ebrahimi,
357
M., β-lactoglobulin and α-lactalbumin Hydrolysates as Sources of Antibacterial
358
Peptides. J. Agric. Sci. Tech.-Iran 2014, 16, 1587-1600.
359
[5] Svensson, M.; Sabharwal, H.; Håkansson, A.; Mossberg, A.; Lipniunas, P.; Leffler,
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
360
H.; Svanborg, C.; Linse, S., Molecular characterization of α–lactalbumin folding
361
variants that induce apoptosis in tumor cells. J. Biol. Chem. 1999, 274(10),
362
6388-6396.
363
[6] Sadat, L.; Cakir-Kiefer, C.; N Negue, M.; Gaillard, J.; Girardet, J.; Miclo, L.,
364
Isolation and identification of antioxidative peptides from bovine α-lactalbumin. Int.
365
Dairy J. 2011, 21(4), 214-221.
366 367
[7] Maynard, F.; Jost, R.; Wal, J., Human IgE binding capacity of tryptic peptides from bovine α-lactalbumin. Int. Arch. Allergy Imm. 1997, 113(4), 478-488.
368
[8] Bu, G.; Luo, Y.; Zheng, Z.; Zheng, H., Effect of heat treatment on the antigenicity
369
of bovine α-lactalbumin and β-lactoglobulin in whey protein isolate. Food Agric.
370
Immunol. 2009, 20(3), 195-206.
371
[9] Tammineedi, C. V.; Choudhary, R.; Perez-Alvarado, G. C.; Watson, D. G.,
372
Determining the effect of UV-C, high intensity ultrasound and nonthermal
373
atmospheric plasma treatments on reducing the allergenicity of α-casein and whey
374
proteins. LWT-Food Sci. Technol. 2013, 54(1), 35-41.
375
[10] Enomoto, H.; Hayashi, Y.; Li, C. P.; Ohki, S.; Ohtomo, H.; Shiokawa, M.; Aoki,
376
T., Glycation and phosphorylation of α-lactalbumin by dry heating: Effect on
377
protein structure and physiological functions. J. Dairy Sci. 2009, 92(7), 3057-3068.
378
[11] Jiang, Z.; Brodkorb, A., Structure and antioxidant activity of Maillard reaction
379
products from α-lactalbumin and β-lactoglobulin with ribose in an aqueous model
380
system. Food Chem. 2012, 133(3), 960-968.
381
[12] Ter Haar, R.; Westphal, Y.; Wierenga, P. A.; Schols, H. A.; Gruppen, H.,
ACS Paragon Plus Environment
Page 18 of 34
Page 19 of 34
Journal of Agricultural and Food Chemistry
382
Cross-linking behavior and foaming properties of bovine α-lactalbumin after
383
glycation with various saccharides. J. Agric. Food Chem. 2011, 59(23),
384
12460-12466.
385
[13] Li, Z.; Luo, Y.; Feng, L., Effects of Maillard reaction conditions on the
386
antigenicity of α-lactalbumin and β-lactoglobulin in whey protein conjugated with
387
maltose. Eur. Food Res. Technol. 2011, 233(3), 387-394.
388
[14] Bu, G.; Lu, J.; Zheng, Z.; Luo, Y., Influence of Maillard reaction conditions on
389
the antigenicity of bovine α‐lactalbumin using response surface methodology. J.
390
Sci. Food Agric. 2009, 89(14), 2428-2434.
391
[15] Nacka, F.; Chobert, J.; Burova, T.; Léonil, J.; Haertlé, T., Induction of new
392
physicochemical and functional properties by the glycosylation of whey proteins. J.
393
Protein Chem. 1998, 17(5), 495-503.
394
[16] Zhang, Q.; Tu, Z.; Wang, H.; Huang, X.; Shi, Y.; Sha, X.; Xiao, H., Improved
395
glycation after ultrasonic pretreatment revealed by high-performance liquid
396
chromatography–linear Ion trap/orbitrap high-resolution mass spectrometry. J.
397
Agric. Food Chem. 2014, 62(12), 2522-2530.
398 399
[17] Soria, A. C.; Villamiel, M., Effect of ultrasound on the technological properties and bioactivity of food: a review. Trends Food Sci. Tech. 2010, 21(7), 323-331.
400
[18] Legay, M.; Gondrexon, N.; Le Person, S.; Boldo, P.; Bontemps, A.,
401
Enhancement of heat transfer by ultrasound: review and recent advances. Int. J.
402
Chem. Eng. 2011, 2011.
403
[19] Jambrak, A. R.; Mason, T. J.; Lelas, V.; Krešić, G., Ultrasonic effect on
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
404
physicochemical and functional properties of α-lactalbumin. LWT-Food Sci.
405
Technol. 2010, 43(2), 254-262.
406
[20] Chandrapala, J.; Zisu, B.; Kentish, S.; Ashokkumar, M., The effects of
407
high-intensity ultrasound on the structural and functional properties of
408
α-Lactalbumin, β-Lactoglobulin and their mixtures. Food Res. Int. 2012, 48(2),
409
940-943.
410
[21] Zhang, M.; Zheng, J.; Ge, K.; Zhang, H.; Fang, B.; Jiang, L.; Guo, H.; Ding, Q.;
411
Ren, F., Glycation of α-lactalbumin with different size saccharides: Effect on
412
protein structure and antigenicity. Int. Dairy J. 2014, 34(2), 220-228.
413
[22] Chen, Y.; Tu, Z.; Wang, H.; Zhang, Q.; Zhang, L.; Sha, X.; Huang, T.; Ma, D.;
414
Pang, J.; Yang, P., The Reduction in the IgE-Binding Ability of β-Lactoglobulin by
415
Dynamic High-Pressure Microfluidization Coupled with Glycation Treatment
416
Revealed by High-Resolution Mass Spectrometry. J. Agric. Food Chem. 2017,
417
65(30), 6179-6187.
418
[23] Liu, J.; Tu, Z.; Shao, Y.; Wang, H.; Liu, G.; Sha, X. M.; Zhang, L.; Yang, P.,
419
Improved antioxidant activity and glycation of α-lactalbumin after ultrasonic
420
pretreatment revealed by high-resolution mass spectrometry. J. Agric. Food Chem.
421
2017, 65(47), 10317-10324.
422
[24] Chen, Y.; Liang, L.; Liu, X.; Labuza, T. P.; Zhou, P., Effect of fructose and
423
glucose on glycation of β-lactoglobulin in an intermediate-moisture food model
424
system: analysis by liquid chromatography–mass spectrometry (LC–MS) and
425
data-independent acquisition LC–MS (LC–MSE). J. Agric. Food Chem. 2012,
ACS Paragon Plus Environment
Page 20 of 34
Page 21 of 34
Journal of Agricultural and Food Chemistry
426
60(42), 10674-10682.
427
[25] Arita, K.; Babiker, E. E.; Azakami, H.; Kato, A., Effect of chemical and genetic
428
attachment of polysaccharides to proteins on the production of IgG and IgE. J.
429
Agric. Food Chem. 2001, 49(4), 2030-2036.
430
[26] Yang, W.; Tu, Z.; Wang, H.; Zhang, L.; Xu, S.; Niu, C.; Yao, H.; Kaltashov, I.
431
A., Mechanism of the reduction in the IgG and IgE binding of β-lactoglobulin
432
induced by ultrasound pretreatment combined with dry-state glycation: a study
433
using conventional spectrometry and high resolution mass spectrometry. J. Agric.
434
Food Chem. 2017, 97(9), 2714-2720.
435
[27] Li, C.; Xue, H.; Chen, Z.; Ding, Q.; Wang, X., Comparative studies on the
436
physicochemical properties of peanut protein isolate–polysaccharide conjugates
437
prepared by ultrasonic treatment or classical heating. Food Res. Int. 2014, 57, 1-7.
438
[28] Zhang, B.; Chi, Y. J.; Li, B., Effect of ultrasound treatment on the wet heating
439
Maillard reaction between β-conglycinin and maltodextrin and on the emulsifying
440
properties of conjugates. Eur. Food Res. Technol. 2014, 238(1), 129-138.
441
[29] Sun, Y.; Hayakawa, S.; Ogawa, M.; Izumori, K., Evaluation of the site specific
442
protein glycation and antioxidant capacity of rare sugar− protein/peptide conjugates.
443
J. Agric. Food Chem. 2005, 53(26), 10205-10212.
444
[30] Järvinen, K.; Chatchatee, P.; Bardina, L.; Beyer, K.; Sampson, H. A., IgE and
445
IgG binding epitopes on α-lactalbumin and β-lactoglobulin in cow’s milk allergy.
446
Int. Arch. Allergy Imm. 2001, 126(2), 111-118.
447
[31] Shriver, S. K.; Yang, W. W., Thermal and nonthermal methods for food allergen
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
448 449 450
control. Food Eng. Rev. 2011, 3(1), 26-43. [32] Suryanarayana, C. V., Propagation of ultrasonic waves in liquids: a new model. Ultrasonics 1992, 30(2), 104-106.
451
[33] Morisawa, Y.; Kitamura, A.; Ujihara, T.; Zushi, N.; Kuzume, K.; Shimanouchi,
452
Y.; Tamura, S.; Wakiguchi, H.; Saito, H.; Matsumoto, K., Effect of heat treatment
453
and enzymatic digestion on the B cell epitopes of cow's milk proteins. Clin. Exp.
454
Allergy 2009, 39(6), 918-925.
455
ACS Paragon Plus Environment
Page 22 of 34
Page 23 of 34
Journal of Agricultural and Food Chemistry
Figure captions Fig. 1: Schematic depiction of the sample preparation. Fig. 2: The IgE (A) and IgG (B) binding ability of the treated α-LA was performed by inhibition ELISA. IC50: the concentration of inhibitors that causes a 50% inhibition of antibody binding (μg/mL). Pooled rabbit anti-α-LA-sera or Anti-α-LA patients' pooled sera (50 μL/well) were incubated separately with 0.5, 1, 5, 30, 60, 100 μg/mL (50 μL/well) of glycated α-LA as inhibitors. Fig. 3: MALDI-TOF-MS analysis of N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LAG-90 and U-LA-G-150. Fig. 4: Mass spectra for the unglycated peptides of U-(LA-G)-150. (A) peptide 9-23 at +
+
m/z 561.31393 , (B) peptide 10-23 at m/z 512.29063 , (C) peptide 53-71 at m/z 587.02334+, (D) peptide 92-103 at m/z 463.94373+, (E) peptide 104-117 at m/z 566.95903+. The determined peptides are labelled by residue numbers. The m/z differences between glycated and unglycated peptides are indicated above the arrows. Fig. 5: The ETD MS/MS spectra of the glycated peptides. (A) the glycated peptide 923 (FRELKDLKGYGGVSL) with m/z of 615.33143+, (B) the glycated peptide 10-23 +
(RELKDLKGYGGVSL) with m/z of 566.30823 , (C) the glycated peptide 53-71 (FQINNKIWCKDDQNPHSSN) with m/z of 668.04974+, (D) the glycated peptide 91103 (VKKILDKVGINY) with m/z of 428.73764+, (E) the glycated peptide 92-103 (VKKILDKVGINY) with m/z of 571.98863+, (F) the glycated peptide 104-117 (WLAHKALCSEKLDQ) with m/z of 620.97673+. The sequence of per peptide is depicted on the top of the spectrum. The identified glycated sites are indicated by a
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
line with galactose. The c and z ions are shown by the numbers and lines. Fig. 6: Ribbon diagram of the glycated α-LA (PDB 1F6S). The glycation sites are colored as follows: grey, framework of α-LA; red, glycation sites of the glycated αLA; green, additional glycation sites of the glycated α-LA after ultrasonication. Fig. 7: The average degree of substitution per peptide molecule (DSP) value of glycated sites of N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LA-G-90 and U-LA-G150.
ACS Paragon Plus Environment
Page 24 of 34
Page 25 of 34
Journal of Agricultural and Food Chemistry
Fig. 1: Schematic depiction of the sample preparation.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 26 of 34
Fig. 2: The IgE (A) and IgG (B) binding ability of the treated α-LA was performed by inhibition ELISA. IC50: the concentration of inhibitors that causes a 50% inhibition of antibody binding (μg/mL). Anti-α-LA rabbit pooled sera or Anti-α-LA patients' pooled sera (50 μL/well) were incubated separately with 0.5, 1, 5, 30, 60, 100 μg/mL (50 μL/well) of the corresponding glycated α-LA as inhibitors.
100
A
90
Inhibition (1-B/B0)*100
80 70
N-LA N-LA-G U-(LA-G)-90 U-(LA-G)-150 U-LA-G-90 U-LA-G-150
12.2
60
IC50
50
4.5
11.1
40
14.2 16.2 14.5
30 20 10 0 -0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Log(inhibitor concentration)
100
B
90
Inhibitor (1-B/B0)*100
80 70 60 50
N-LA N-LA-G U-(LA-G)-90 U-(LA-G)-150 U-LA-G-90 U-LA-G-150
3.9
1.1
IC50
4.6
5.1 6.4
5.5
40 30 20 10 0 -0.5
0.0
0.5
1.0
1.5
2.0
Log(inhibitor concentration)
ACS Paragon Plus Environment
2.5
3.0
Page 27 of 34
Journal of Agricultural and Food Chemistry
Fig. 3: MALDI-TOF-MS analysis of N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LAG-90, U-LA-G-150.
100 100
100
U-(LA-G)-90
U-(LA-G)-150
15309.74
N-LA-G 14987.07
80
80
15284.94
40
60
40
20000
25000
30000
35000
0 10000
40000
15000
20000
25000
mass (m/z) 100
40
30000
35000
40000
0 10000
15000
20000
100
U-LA-G-90
15153.45
U-LA-G-150 15168.48
80
80
60
40
0 10000
60
40
20
20
15000
20000
25000
mass (m/z)
30000
35000
40000
0 10000
25000
mass (m/z)
mass (m/z)
% intensity
15000
60
20
20
20
0 10000
% intensity
% intensity
60
% intensity
% intensity
80
15000
20000
25000
30000
mass (m/z)
ACS Paragon Plus Environment
35000
40000
30000
35000
40000
Journal of Agricultural and Food Chemistry
Page 28 of 34
Fig. 4: Mass spectra for the unglycated peptides of U-(LA-G)-150. (A) peptide 9-23 at m/z 561.31393+, (B) peptide 10-23 at m/z 512.29063+, (C) peptide 53-71 at m/z 587.02334+, (D) peptide 92-103 at m/z 463.94373+, (E) peptide 104-117 at m/z 566.95903+ The determined peptides are labelled by residue numbers. The m/z differences between glycated and unglycated peptides are indicated above the arrows.
A
100
AA (9-23)
100
m/z=54.0175
20
0 550
560
570
580
590
600
+3 615.3314
610
620
60
40
m/z=54.0176 +3 512.2906
20
630
640
0 480
650
490
500
510
520
mass (m/z) 100
+3 566.3082
530
100
D
80
20
550
560
570
40
+4 627.5363
+4 587.0233
+3 571.9886 +3 517.9707 +3 m/z=54.027 463.9437
m/z=54.0179
m/z=40.513
580
0 560 570 580 590 600 610 620 630 640 650 660 670 680
mass (m/z) AA (104-117)
E
60
40
m/z=54.0177 20
0 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
mass (m/z)
0 550
+3 566.959
560
570
580
+4 668.0497
m/z=40.5134
20
80
60
40
540
60
mass (m/z) AA (92-103)
Relative abundance
+3 561.3139
AA (53-71)
C
80
relative abundance
Relative abundance
60
40
100
80
Relative abundance
Relative abundance
80
AA (10-23)
B
590
600
610
mass (m/z)
ACS Paragon Plus Environment
+3 620.9767
620
630
640
650
Page 29 of 34
Journal of Agricultural and Food Chemistry
Fig. 5: The ETD MS/MS spectra of the glycated peptides. (A) the glycated peptide 923 (FRELKDLKGYGGVSL) with m/z of 615.33143+, (B) the glycated peptide 10-23 (RELKDLKGYGGVSL) with m/z of 566.30823+, (C) the glycated peptide 53-71 (FQINNKIWCKDDQNPHSSN) with m/z of 668.04974+, (D) the glycated peptide 91-103 (VKKILDKVGINY) with m/z of 428.73764+, (E) the glycated peptide 92-103 (VKKILDKVGINY) with m/z of 571.98863+, (F) the glycated peptide 104-117 (WLAHKALCSEKLDQ) with m/z of 620.97673+. The sequence of per peptide is depicted on the top of the spectrum. The identified glycated sites are indicated by a line with galactose. The c and z ions are shown by the numbers and lines. AA (9-23) C
2 3 4 5 6 7 8 9 10 11 12 13 14 15
AA (10-23)
F R E L K D LK GY G GX S L
A
15 14 13 12
100
c
B
Z
10 9 8 7 6 5 4 3 2
Gal
926.4910,z8
1039.5513,z9 1062.5466,c7 1119.6566,c8 1154.5414,z10 1282.7168,c9,z11 1339.7493,c10 1396.7416,c11,z+1 12 1495.7174,c12 1524.7667,z13 1582.8691,c13
772.5216,c6
544.4542,c4 579.2954,z6 636.8537,z7 659.4356,c5
40
303.1801,c2 360.2628,z+1 4 416.4059,c3,z5
Relative abundance
1730.8760,c+1 14
1681.9294,z+1 14
60
20
0
0 600
800
200
1000 1200 1400 1600 1800 2000
400
600
800
1000
AA (53-71)
20
100 Z
Gal 80
0
1400
1600
1800
AA (91-103) D
c
60
40
20
2 3 4 5
6 7
8 9 10 11 12 13
C VKK I LDK VG I NY 13 12 11 10 9
8 7
5
4 3
2
Gal 1067.5203,z8
639.4443,z6
3 2
Relative abundance
40
767.4174,z7
60
293.3460,c2 406.3763,c3 428.3430,z4
Relative abundance
80
634.5757,c5
520.4766,c4
Gal
9 8 7 6 5 4
1633.7390,z12 1673.3113,c10 1747.7017,z+1 13 1789.8377,c+1 11 1903.8579,c12
924.6260,c6 997.4681,z9 1016.8621,c132+ 2+ 1037.6895,c7 1076.3084,z15 1122.3626,c152+ 2+ 1190.4365,z17 ,c162+ 1223.7468,c8 1234.7122,c172+ 1254.7854,z182+ 2+ 1289.7769,z+1 10 1278.7280,c18 1383.7105,c9 1447.7351,z11
13 12 11
759.5822,c6 838.5655,z6 874.5732,c7 954.4459,z7
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
646.4925,c5
2 3
R Q I N N K I WC KD DQ N P H S S N 19 18 17 16 15
277.1972,c2 280.0900,z2 393.2735,z3 405.3481,c3 450.3312,z4 549.4022,z5 533.4385,c4
C
C
1200
m/z
m/z
1309.7411,z10+1
400
1321.7047,c10+1 1433.8119,c11 1437.8254,z11+1 1536.8190,z12+1
200
100
7 6 5 4 3 2
14 13121110 9
80
174.1142,c1
20
764.4419,z8 853.5383,c5 877.5728,z9 968.4913,c6 992.5053,z10 1081.5833,c7 1209.6196,c8
321.2676,c2 450.2798,c3
563.3777.c4 638.9337,z7
40
416.1037,z5
Relative abundance
60
1226.5387,c9 1282.7128,z11 1395.7565,z12 1429.7710,c10 1486.7706,c11 1524.7389,z13 1543.8335,c12 1643.8618,c+1 13
Gal 80
2 3 4 5 6 7 8 9 10 1112 13 14
R E L K D L KGYGGVS L
1164.6488,c8 1180.5967,z9 1263.7186,c9
100
1200
1400
0 200
400
600
800
1000 1200 1400 1600 1800 2000
200
400
600
800
m/z
1000
m/z
ACS Paragon Plus Environment
1600
1800
z
z
40
20
200 400 600 800 1000 1200
Gal
0 1400 1600 1800 60
40
246.3444,z2
100
z
80
20
0 200 400 600
815.5446,c5 886.6234,c6 866.7630,z7 977.4742,z8 999.6346,c7 1048.4681,z9 1160.5671,c8 1247.6023,c9 1338.6513,z10 1376.6639,c10 1475.6777,z11 1504.7372,c11 1546.7170,z12 1617.8076,c12 1659.8136,z13 1732.0287,c13
AA (92-103) 359.3906,z3 388.4573,c3 487.4854,z4 525.4684,c4 617.4882,z+1 5 703.4441,z6
2 3 4 5 6 7 8 9 10 11 12
204.3369,c1
V K K I LD K V G I N Y
317.4932,c2
5 4 3 2
Relative abundance
10 9 8 7
1598.8585,z11
Gal 1323.7513,c+1 9
12
1435.7590,c10
80
1549.9285,c11
c
1166.1193,c7 1180.7336,z9 1265.7496,c8 1308.6963,z10
E
1067.6547,z8
761.6832,c5 839.3754,z6 876.7221,c6 954.3035,z7
407.4978,c2
60
451.5798,z+1 4 535.3893,c3 549.6426,z5 648.4789,c4
100
393.6325,z3
Relative abundance
Journal of Agricultural and Food Chemistry
F C
m/z 800
m/z
ACS Paragon Plus Environment
Page 30 of 34
W L AH K AL CS E K LD Q
2 3 4 5 6 7 8 9 10 11 12 13 14
AA (104-117)
14 13 12 11
Gal
9 8 7 6 5 4 3 2
Z
0 1000 1200 1400 1600 1800 2000
Page 31 of 34
Journal of Agricultural and Food Chemistry
Fig. 6: Ribbon diagram of the glycated α-LA (PDB 1F6S). The glycation sites are colored as follows: grey, framework of α-LA; red, glycation sites of the glycated αLA; green, additional glycation sites of the glycated α-LA after ultrasonication.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 32 of 34
Fig. 7: The average degree of substitution per peptide molecule (DSP) value of glycated sites of N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LAG-90 and U-LA-G-150. N-LA-G U-(LA-G)-90 U-(LA-G)-150 U-LA-G-90 U-LA-G-150
1.0
0.8
DSP
0.6
0.4
0.2
0.0 9-23
10-23
53-71
92-103
peptide
ACS Paragon Plus Environment
104-117
Page 33 of 34
Journal of Agricultural and Food Chemistry
Table 1. Summary of the glycated peptides in the N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LA-G-90 and U-LA-G-150. Sample N-LA-G
Peptide
m/z
Δm
location
Glycated peptide
ppm
9-23
615.33053+
-1.68
10-23
566.30903+
91-103
428.73774+
92-103
571.98903+
104-117
Sequencea
Glycated
(V)FRELKDLKGYGGVSL(P)
K13
0.53
(F)RELKDLKGYGGVSL(P)
K16
-0.23
(M)C*VKKILDKVGINY(W)
K98
-6.01
(C)VKKILDKVGINY(W)
K93, K98
465.98484+
0.48
(Y)WLAHKALC*SEKLDQ(W)
K108 K13
site
U-(LA-G)-90 9-23
615.33143+
-0.16
(V)FRELKDLKGYGGVSL(P)
10-23
566.30873+
0.06
(F)RELKDLKGYGGVSL(P)
K16
53-71
668.04964+
-0.68
(L)FQINNKIWC*KDDQNPHSSN(I)
K58, K62
91-103
428.73804+
0.53
(M)C*VKKILDKVGINY(W)
K98
92-103
571.98893+
-6.13
(C)VKKILDKVGINY(W)
K93, K98
104-117
620.97723+
0.91
(Y)WLAHKALC*SEKLDQ(W)
K108 K13
U-(LA-G)-150 9-23
615.33143+
-1.52
(V)FRELKDLKGYGGVSL(P)
10-23
566.30823+
0.71
(F)RELKDLKGYGGVSL(P)
K16
53-71
668.04974+
-1.04
(L)FQINNKIWC*KDDQNPHSSN(I)
K58, K62
91-103
428.73764+
-0.47
(M)C*VKKILDKVGINY(W)
K98
92-103
571.98863+
-6.13
(C)VKKILDKVGINY(W)
K93, K98
104-117
620.97673+
1.07
(Y)WLAHKALC*SEKLDQ(W)
K108
9-23
461.75074+
0.49
(V)FRELKDLKGYGGVSL(P)
K13
10-23
566.30923+
0.88
(F)RELKDLKGYGGVSL(P)
K16
53-71
627.53694+
0.40
(L)FQINNKIWC*KDDQNPHSSN(I)
K62
91-103
571.31543+
1.34
(M)C*VKKILDKVGINY(W)
K98
92-103
571.98933+
-5.60
(C)VKKILDKVGINY(W)
K93, K98
104-117
465.98364+
-1.61
(Y)WLAHKALC*SEKLDQ(W)
K108
9-23
615.33153+
-0.05
(V)FRELKDLKGYGGVSL(P)
K13
10-23
566.30873+
-0.12
(F)RELKDLKGYGGVSL(P)
K16
53-71
627.53684+
0.20
(L)FQINNKIWC*KDDQNPHSSN(I)
K62
91-103
428.73784+
0.18
(M)C*VKKILDKVGINY(W)
K98
92-103
571.98883+
-6.36
(C)VKKILDKVGINY(W)
K93, K98
104-117
465.98484+
0.56
(Y)WLAHKALC*SEKLDQ(W)
K108
U-LA-G-90
U-LA-G-150
aC*
refers to carbamidomethyl.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 34 of 34
Graphical abstract
Native α-LA Dry-heating glycation with galactose
Ultrasonic pretreatment D-(+)-Galactose
Ultrasonic pretreatment Dry-heating Lys 108
Lys 108 Lys 16
Lys 13
Lys 16 Lys 98 Lys 93
Dry-heating glycation with galactose
Lys 13
Lys 108 Lys 16
Lys 98 Lys 93 Lys 58
Lys 62 Glycation extent ACS Paragon Plus Environment IgE/IgG binding ability
Lys 13
Lys 98 Lys 93
Lys 62