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Improved antioxidant activity and glycation of #-lactalbumin after ultrasonic pretreatment revealed by high-resolution mass spectrometry Jun Liu, Zong-cai Tu, Yan-hong Shao, Hui Wang, Guang-xian Liu, Xiao-mei Sha, Lu Zhang, and Ping Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03920 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 6, 2017
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Journal of Agricultural and Food Chemistry
Improved antioxidant activity and glycation of α-lactalbumin after ultrasonic pretreatment revealed by high-resolution mass spectrometry
1 2 3 4 5 6 7 8
Jun Liu*, Zong-cai Tu*, †, ‡, Yan-hong Shao*, Hui Wang†, ‡, Guang-xian Liu*, Xiao-mei
9
Sha*, Lu Zhang*, Ping Yang*
10 11 12 13
*
14
Normal University, Nanchang, Jiangxi 330022, China;
15
†
16
Nanchang, Jiangxi 330047, China;
College of Chemistry and Chemical Engineering, College of Life Science, Jiangxi
State Key Laboratory of Food Science and Technology, Nanchang University,
17 18 19
‡
20
E-mail:
21
(Hui-Wang)
Corresponding authors. Tel.: +86-791-8812-1868; fax: +86-791-8830-5938.
[email protected] (Zong-cai
Tu),
[email protected] 22 23 24 25 26 27 28 29 30
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Abstract: High-resolution mass spectrometry was performed to investigate the
32
relationship between bovine α-lactalbumin (α-LA) subjected to ultrasonication and
33
glycation treatment with respect to antioxidant activity. After α-LA was pretreated by
34
ultrasonication combined with glycation, the treated α-LA showed low intrinsic
35
fluorescence emission and high antioxidant activity at increased ultrasonic power
36
levels. Prior to ultrasonic pretreatment, three glycated sites were identified, whereas
37
the number of glycation sites was increased to four, four, five and six after ultrasonic
38
power at 60, 90, 120, and 150 W/cm2, respectively, for 15 min. Thus, no obvious
39
difference was found among the glycation sites at the ultrasonic power of 60 and 90
40
W/cm2. The average degree of substitution per peptide molecule of α-LA was used to
41
evaluate the glycation level for per glycation site. All the samples pretreated by
42
ultrasonication exhibited a higher glycation level compared with the untreated
43
samples. Ultrasonic power at 150 W/cm2 showed the most highly enhanced glycation
44
extent and antioxidant activity. Therefore, the intensified glycation extent and the
45
conformational changes of protein were responsible for the increase of antioxidant
46
activity of α-LA. Moreover, high-resolution mass spectrometry is an efficient
47
technique to understand the mechanism of the improved antioxidant activity.
48 49 50
Keywords: α-lactalbumin, ultrasonication, glycation, antioxidant activity, mass
51
spectrometry
52 53 54 55
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ABBREVIATION USED
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α-LA, alpha-lactalbumin; Glc, glucose; DSP, the average degree of substitution per
58
peptide molecule; MRPs, Maillard reaction products; PBS, phosphate buffer solution;
59
AA, amino acid; TFA, trifluoroacetic acid; DTT, DL-dithiothreitol; ABTS,
60
2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate);
61
preparation; SEC, size exclusion chromatography; HPLC, high performance liquid
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chromatography; ETD MS/MS, electron transfer dissociation mass spectrometry/mass
63
spectrometry.
FASP,
64 65 66 67 68 69 70 71 72 73 74 75 76 77
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filter-aided
sample
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Introduction
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Small bovine protein α-lactalbumin (α-LA), is suited as an ingredient for infant
81
nutrition in the food1, which consists of 123 amino acid residues and four disulfide
82
bridges, and has molecular weight around 14.2 KDa2. α-LA is a simple model Ca2+
83
binding protein and has immune-modulating3, antioxidant4, antibacterial5 or antitumor
84
activity6, 7. Its hydrolyzate also exhibit antioxidant activity8. Reported strategies, such
85
as heat treatment9, 10, glycation11 and high-intensity ultrasonic treatment12 nationally
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modulate α-LA functionality and thus address physical and chemical pathways. For
87
these processes, glycation is the first step of Maillard reaction occurring between
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amino and carbonyl compounds, and plays an important role in food processing,
89
especially in protein modification13. Moreover, α-LA is supplemented to infant
90
formulae which undergo the glycation reaction, then may modulate its functionality14.
91
The weight ratio of protein to sugar, reaction temperature and time; pH, water activity;
92
and reactant structure are the major factors that influence the glycation reaction; these
93
factors affect the glycation reaction between α-LA and maltose and reduces the
94
antigenicity of α-LA15. In addition, Velusamy et al. studied the influence of glucose
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and pH on the glycation of α-LA and found that glucose and pH affect the
96
stabilization of α-LA16. Although glycation is widely used to improve functional
97
properties of α-LA13, 17, a single modification alone cannot improve the functional
98
properties to a satisfactory result.
99
High-intensity ultrasonication, an emerging nonthermal technology, presents a
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great deal of successful improvements on the functional properties of food in four
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ways, including heating effects, acoustic cavitation, acoustic streaming, and fluid
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particle oscillations18,
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glycation of bovine serum albumin (BSA)20 and unfold the structure of ovalbumin21.
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Ultrasonication and glycation can disrupt the secondary and tertiary structure of
105
α-LA22, and glycation can greatly improve the antioxidant activity8. Glycation occurs
106
on the Lys and Arg of a protein and alters the peptides. Thus, the conformational
107
changes of α-LA owing to ultrasonic pretreatment coupled with glycation were
108
associated with the antioxidant activity. Moreover, the glycation extent is also
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important for the explanation of the functional properties of glycoprotein. Glycation
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extent was evaluated mostly using traditional methods and parameters, such as free
111
amino acid content, browning intensity and fluorescence intensity etc15, 23. However,
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in these methods, the glycation sites and glycation extent at each site in the protein
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cannot be identified because the change in glycation degree are determined at the
114
protein level. Mass spectrometry is widely used to analyze the nature and extent of
115
protein modification24, 25, and exactly investigate the processes inside the protein. To
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date, although the mechanisms by which proteins and Maillard conjugates exert their
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antioxidant activity are poorly understood26, the influence of ultrasonic pretreatment
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combined with glycation on the antioxidant activity and glycation extent of α-LA has
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not been studied. Providing a method of protein modification in the food industry also
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requires investigating its antioxidant activity.
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19
. In our previous work, ultrasonication can promote the
The overall goal of this research was to study the impact of ultrasonic power on
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the antioxidant activity and glycation extent of α-LA treated by glycation. We firstly
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applied the ultrasonic technique to the structural perturbation of α-LA. Ultrasonicated
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α-LA was then subjected to glycation. The antioxidant activity of treated α-LA was
125
evaluated by the ABTS radical-scavenging activity. The glycation sites and extent of
126
α-LA were further examined by high-resolution mass spectrometry. The results of this
127
research enhance our understanding of the relationship between antioxidant activity
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and structural changes of α-LA induced by ultrasonic pretreatment combined with
129
glycation.
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Materials and methods
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Chemicals and materials
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Alpha-lactalbumin (α-LA) from bovine milk (L6010, Type Ⅲ, ≥ 85%), Glucose
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(G8270), pepsin (P6887, 3,200-4,500 units/mg protein) were purchased from Sigma
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Chemical Co. (St. Louis, MO, USA); DL-1,4-Dithiothreitol (AC165680050) was
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purchased from Fisher Scientific Inc. (Waltham, MA, USA). All other reagents and
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solvents used were of analytical and high performance liquid chromatography (HPLC)
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grade.
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Sample Preparation
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Native α-LA (100 mg) was dissolved in 100 mL of 50 mM phosphate buffer saline
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(PBS) at pH 7.4. Ten milliliters of α-LA were split into 25 mL flat bottom conical
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flasks, and immersed in ice bath. The solution was treated by probe sonicator
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(Misonix Qsonica Q700 Sonicator, USA, 20 kHz) equipped with a microtip probe
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(1/8 in. = 3 mm) with a 9s on and 1s off pulsation at an actual ultrasonic intensity of 0,
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60, 90, 120 and 150 W/cm2 for 15 min, respectively, to ensure the temperature of the
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sample solution is not elevated (lower than 15 oC).
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In case of glycation, 1.0 mg of glucose (Glc) was added to 1.0 mL of the native
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α-LA and ultrasonicated α-LA solution, separately. After lyophilization, the powders
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of native BSA, native α-LA-glucose, ultrasonicated α-LA-glucose complex were
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incubated at 65% relative humidity (saturated potassium iodide solution) and 55 oC
150
for 3 h. The reaction was stopped by transferring the sample tubes into an ice bath.
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The free glucose and salts were filtered by a Centricon centrifugal filter unit (3000 Da
152
cutoffs, Millipore, Bedford, MA, USA). The concentration of all the samples were
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adjusted to 10 mg/mL and stored at 4 oC for further analyses. Native α-LA was named
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N-LA. The glycated samples with ultrasonic pretreatment at 0, 60, 90, 120, and 150
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W/cm2 for 15 min were named N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120,
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LA-Glc-150, respectively. The treatments were performed in triplicates.
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Size exclusion chromatography
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Sample solution (30 uL) were purified by size exclusion chromatography (SEC) on a
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TSK Gel 3000 SWXL column (TOSOH Bioscience, King of Prussia, PA, USA) using
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an Agilent 1100 (Agilent Technologies, Palo Alto, CA, USA) HPLC system. The
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separation was performed with 0.1 M ammonium acetate (pH 6.8) at a flow rate of 0.5
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mL/min and monitored with 280 nm ultraviolet (UV) detection.
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Intrinsic fluorescence emission spectroscopy
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1.0 mg/mL of samples were prepared with PBS (50 mM, pH 7.4). The intrinsic
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fluorescence emission spectra were obtained by a Hitachi F-7000 fluorescence
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spectrophotometer (Hitachi, Ltd, Tokyo, Japan). The emission spectra were recorded
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from 300 nm to 400 nm (both at a constant slit of 5 nm) with excitation at 290 nm and
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PBS was used as blank.
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Determination of ABTS+ radical scavenging activity
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The ABTS+ radical scavenging activity was estimated using a previously reported
171
method27. The ABTS+ was formed by adding K2S2O8 to ABTS. In brief, 20 µL of each
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protein sample (2, 4, 6, 8, and 10 mg/mL) was mixed with the diluted ABTS+ solution
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(absorbance of 0.70 ± 0.01 at 734 nm). The mixture was incubated in the dark for 6
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min, and the absorbance of mixture was measured at 734 nm using a SynergyTM HT
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Multi-Mode Microplate Reader (BioTek Instruments Co. Ltd., VT, USA). The ABTS+
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radical scavenging activity (%) was calculated:
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ABTS radical scavenging activity (%) =
!"#
× 100%
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where Acontrol is the absorbance of the control (ABTS+ solution without samples) and
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Asample is the absorbance of the samples.
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Filter-aided sample preparation (FASP)
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Protein digestion was conducted using to the FASP method according to the method
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of Chen et al.25 with slight modifications.10 µL samples (10 mg/mL) were mixed with
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200 µL of 150 mM urea (UA) buffer (8 M urea, 150 mM Tris-HCl pH 8.0) and 20 µL
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DTT (100 mM) and incubated at 95 oC for 5 min, then cooled in the ice bath. The
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mixture was transferred to a Centricon centrifugal filter unit (10000 Da cutoffs,
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Millipore, Bedford, MA, USA). The samples were centrifuged at 14000 × g for 15
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min at 25 oC, 200 µL UA buffer was then added and centrifuged at 14000 × g for 15
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min. After, 100 µL iodoacetamide (50 mM) in UA buffer was added and incubated at
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room temperature in the dark for 60 min, and then centrifuged at 14000 × g for 10
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min. The filters were washed thrice using UA buffer (100 µL), followed by the
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additional of 100 µL ammonium bicarbonate solution (50 mM, pH 8.5), then
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centrifuged at 14000 × g for 10 min and repeated this step thrice. 30 µL pepsin (10
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mg/mL) was added to each filter and incubated 4 oC for 15 min. The reaction was
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then quenched by adding 6 µL of 10% TFA. The filtered solution was collected by
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centrifugation at 14000 × g for 15 min for next step analysis.
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Analysis by HPLC-ETD-MS/MS
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Samples were analyzed on a Thermo Fisher Q Exactive Mass Spectrometer (Thermo
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scientific, Waltham, MA, USA). Solvent A was 0.1% formic acid (FA) in ultrapure
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H2O, whereas solvent B consisted of 84% acetonitrile in H2O, 0.1% FA. For analysis
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150 µL of proteolytic peptides, the digested sample was injected onto a 75 µm i.d. ×
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100 mm 3µm-C18 column (EASY-column, Thermo scientific, Waltham, MA, USA).
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After desalting for 20 min with A, the peptides were eluted at 300 nL/min with a
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gradient of 4-50% B for 40 min, 50-100% B for 5 min, and 100% B for 1 min, then
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analyzed using a Thermo Fisher Q Exactive Mass Spectrometer. Detection mode:
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positive ions. The scan range was set to 150–2000 m/z, and a resolution of 70000 at
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m/z 200 was applied for acquiring survey scans. The dry gas was set to a flow of 7.0
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L/min, a capillary temperature of 250 °C, and a spray voltage of 1.8 KV. The m/z of
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peptides and peptide fragments was detected by the following methods: full
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scan-every time, and then collected 20 fragmentography (MS2 scan).
210 211
To further compare the extent of glycation extent of each peptide, the average degree of substitution per peptide molecule (DSP) of α-LA was calculated: DSP =
∑7.89 i × I+,+-./,0 . × 123456, ∑7.89 I+,+-./,0 . × 123456,
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where I is the sum of the intensities of the glycated peptides, and i is the number of
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glucose units attached to the peptide in each glycated form.
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Database Search
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Original RAW file was performed to Mascot Server (Matrix Science) by proteome
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discoverer, and ETD was used as the method of check database. The following search
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parameters were used: enzyme = no specific, missed cleavage = 5, fixed modification:
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carbamidomethyl (C), variable modification: oxidation (M), glycation (K, R and N).
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Mass values: monoisotopic peptide mass tolerance = ± 10 ppm, MS/MS tolerance = ±
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0.5 Da. The following search database were used: > sp|P00711|LALBA_BOVIN
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Alpha-lactalbumin OS = Bos taurus GN = LALBA PE = 1 SV = 2
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MMSFVSLLLVGILFHATQAEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTS
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GYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTD
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DIMCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL. STQTΑLA. Parent ion
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tolerance = ± 10 ppm, daughter ion tolerance = ± 0.5 Da. Results of filtering
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parameters: Mascot Score ≥ 20.
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Results and Discussion
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Size exclusion chromatography
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Size exclusion chromatography (SEC) separates biomolecules according to their
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molecular size28. Fig. 1 displays SEC diagrams for N-LA, N-LA-Glc, LA-Glc-60,
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LA-Glc-90, LA-Glc-120, and LA-Glc-150. Monomer of N-LA was calculated by SEC,
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after glycation; the monomer of α-LA-Glc conjugates was also observed. However,
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the elution time (monomer) of ultrasonicated α-LA-Glc conjugates shifted to around
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19.5 min, which is much shorter than the elution time of N-LA and N-LA-Glc at 20.6
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min and 19.8 min, respectively. This result indicates the formation of proteinaceous
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high molecular weight components after ultrasonication combined with glycation.
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However, no obvious difference is found in the α-LA-Glc conjugates pretreated by
238
different ultrasonic power levels. This finding may be that the SEC could not reveal
239
their changes exactly and further work needs to be done through mass spectrometry.
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On the other hand, the aggregation of N-LA was also assessed by SEC (Fig. 1). The
241
aggregation of N-LA was not observed at 65% relative humidity (saturated potassium
242
iodide solution) and 55 oC for 3 h, indicating that α-LA was not denatured at current
243
glycation conditions.
244
Intrinsic fluorescence emission spectroscopy
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The intrinsic tryptophan fluorescence emission spectra of all the samples were
246
presented in Fig. 2. When excited at 290 nm, the intensity of N-LA, N-LA-Glc,
247
LA-Glc-60, LA-Glc-90, LA-Glc-120, and LA-Glc-150 were gradually reduced,
248
particularly for LA-Glc-150, which indicates the conformational structure of α-LA
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was dramatically changed by ultrasonic pretreatment combined with glycation, this
250
result can be attributed to the fact that the relatively increased exposure of Trp
251
residues in more hydrophilic surroundings29, solvent relaxation30 and more quenching
252
agents were produced in the conjugates. Furthermore, it may result from the shielding
253
effect of the carbohydrate bound to α-LA31. The α-LA undergoes conformational
254
changes around the Trp residues due to the heating treatment32, as this study used dry
255
heating for glycation. More importantly, the Trp/Tyr residues has the ability to donate
256
a proton. This result may have resulted in their having different antioxidant activities.
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Antioxidant activity
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As shown in the Fig. 3, the ABTS radical-scavenging activity of α-LA significantly
259
improved after glycation to a greater degree compared with that of N-LA (p < 0.05).
260
The enhancement may be due to the reduction of the radical cation because of the
261
reaction between the ABTS radicals and Maillard reaction products (MRPs)27.
262
Furthermore, α-LA undergoes conformational changes around the Trp/Tyr residues
263
because of ultrasonication and glycation (Fig. 2). Antioxidant milk-derived peptides
264
are composed of 5 to 11 amino acids, which include the hydrophobic amino acids Pro,
265
His, Tyr, and Trp14, 33. However, no obvious difference existed between the ABTS
266
radical-scavenging activity of N-LA-Glc and LA-Glc-60 (p > 0.05), but the activity of
267
N-LA-Glc, LA-Glc-90, LA-Glc-120 and LA-Glc-150 were significantly different
268
from one another in (p < 0.05). This finding can be explained by the fact that the
269
compact structure of α-LA could be effectively unfolded at increased ultrasonic
270
power12, thereby accelerating the glycation between α-LA and glucose, producing
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more MRPs, and finally improving the antioxidant activity.
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Glycation Site Determination
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It is known that α-LA has 12 Lys and 1 Arg, including Lys5, Lys13, Lys16, Lys58,
274
Lys62, Lys79, Lys93, Lys94, Lys98, Lys108, Lys114, Lys122 and Arg10, and the
275
N-terminal of Asn45 was linked with a N-acetylglucosamine. Therefore, α-LA
276
contains at least 13 potential glycated sites. Theoretically, if a peptide was
277
mono-glycated by glucose, the corresponding m/z of peaks with 1, 2, 3, 4, or 5
278
charges will display a mass increase of 162.0528 Da, with m/z change of 162.0528,
279
81.0264, 54.0176, 40.5132, and 32.4106 Da, respectively. For the dual-glycated
280
peptides, the mass increase will be 324.1056 Da.
281
As shown in the figure 4, the m/z peaks of the unglycated peptide 61-71, 91-102,
282
104-115, 105-119, and 111-123 were 434.51073+, 462.92253+, 485.93093+, 604.65383+,
283
570.29263+ Da, while the corresponding m/z of glycated peptides were 488.52813+,
284
570.97853+, 539.94843+, 658.67163+and 624.29033+ Da, respectively. The m/z shift of
285
these peaks were 54.0174, 108.056, 54.0175, 54.0178, and 53.9977 Da, respectively,
286
indicating that all these peptides had mono-glycated or dual-glycated peptides.
287
A detailed map of the glycated sites of α-LA was obtained using the ETD
288
MS/MS. The determination of glycated sites Lys62, Lys94, Lys98, Lys108, Lys114
289
and Lys122 by ETD MS/MS is depicted in Fig. 5. The ETD MS/MS spectrum of the
290
mono-glycated
291
488.52813+ exhibited a series of c and z ions (c2−c11 and z2−z11). The glycation site,
292
Lys62, was obtained by the mass difference between the c1 and c3 ions, or between
peptide
61
C(carbamidomethyl)KDDQNPHSSN71
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of
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the z9 and z11, which is the combined mass of Lys and glucose. The glycation sites,
294
Lys98, Lys108, Lys114 and Lys122 were determined according to the ETD MS/MS
295
spectrum of the mono-glycated peptides with m/z values of 428.73784+, 539.94843+,
296
658.67163+, 624.29033+ respectively. A series of c and z ions in the Fig. 5C, D, E, F
297
was
298
91
299
104
300
105
LAHKALC(carbamidomethyl)SEKLDQWL119,
301
111
C(carbamidomethyl)SEKLDQWLC(carbamidomethyl)EKL123
302
Similarly, Fig. 5B shows the mass spectrum of the fragmentation of dual-glycated
303
peptides
304
glycation sites were identified to be Lys94 and Lys98.
detected,
and
matched
well
with
the
fragments
of
peptides
C(carbamidomethyl)VKKILDKVGINY103, WLAHKALC(carbamidomethyl)SEKL115,
91
respectively.
C(carbamidomethyl)VKKILDKVGIN102 with m/z of 570.97853+. The
305
Table 1 shows that the sequence and glycation sites of N-LA-Glc containing
306
three glycation sites, which include Lys62, Lys94, Lys98. After α-LA was pretreated
307
by ultrasonic power at 60 and 90 W/cm2 for 15 min, four sites (Lys62, Lys94, Lys98,
308
Lys108) were glycated. When the sample was ultrasonicated at 120 W/cm2 for 15 min,
309
five sites (Lys62, Lys94, Lys98, Lys108, Lys114) were glycated. Furthermore, six
310
sites (Lys62, Lys94, Lys98, Lys108, Lys114 and Lys122) were found to be glycated
311
in the sample ultrasonicated at 150 W/cm2 for 15 min. The increase of the glycated
312
sites suggested that the α-LA structures loosened under ultrasonic power, which
313
facilitated the glycation reaction and exposed more reactive sites. In this study,
314
Lys114 and Lys122 were not observed in LA-Glc-60 and LA-Glc-90, but they were
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detected in the LA-Glc-120 and LA-Glc-150, suggesting that Lys114 and Lys122
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were glycated with Glc. The glycation proceeded at ultrasonic power levels of 120
317
and 150 W/cm2, which enabled the unfolding of α-LA structure, particularly that of
318
LA-Glc-150. Meanwhile, most glycation sites were measured. High ultrasonic power
319
levels can perturb the protein conformation to a considerably high extent, leading to
320
the intensified glycation. Fig. 6 showed that three additional glycation sites, Lys108,
321
Lys114 and Lys122 were found in the glycated α-LA after ultrasonic pretreatment.
322
Interestingly, the results were consistent with those obtained in our previous studies
323
where the glycated samples with and without ultrasonic pretreatment predominantly
324
occurred on Lys but not on Arg20. Thus, ultrasonic pretreatment can increase the
325
glycation sites by the conformational changes of α-LA.
326
Effects of ultrasonication on the glycation extent of α-LA
327
When α-LA was treated by ultrasonication, the glycation site was significantly
328
increased (Table. 1), indicating that ultrasonication can effectively improve the
329
glycation extent between α-LA and glucose. The DSP values of the glycated peptides,
330
which included 61-71, 91-102, 104-115, 105-119 and 111-123 with ultrasound
331
pretreatment at 0–150 W/cm2, are shown in Fig. 7. The DSP values were significantly
332
promoted by ultrasound pretreatment as a whole. For example, the DSP value of
333
Lys62 was 0.5 in N-LA-Glc. After the sample ultrasonicated at 60–150 W/cm2 for 15
334
min, its DSP further increased to 0.76, 0.79, 0.87 and 0.92. The highest DSP values of
335
all the glycated peptides were found at 150 W/cm2. The peptide, 104-115 was
336
glycated in LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150, its DSP value was
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gradually increased with the increase of ultrasonic power. When ultrasonic power
338
reached to 150 W/cm2, glycated peptide105-119 also had a higher DSP value than
339
LA-Glc-120. Thus, ultrasonication caused the exposure of these regions22, with the
340
more ultrasonic power, the regions will have a better exposure, which then accelerated
341
the glycation and improved the glycation extent of α-LA. This result agreed with the
342
previously performed a similar study on BSA glycation under the influence of
343
ultrasonication20.
344
Mechanism of the increase in the antioxidant activity of α-LA by ultrasonic
345
pretreatment combined with glycation.
346
The extent of Maillard reaction is important for the explanation of the functional
347
behaviors of MRPs. In this study, ultrasonic pretreatment combined with glycation
348
significantly improved the antioxidant activity of α-LA, which was closely related to
349
its structural changes. To explore the mechanism, high resolution mass spectrometry
350
was applied for structural characterization at the molecular level. The results above
351
show that the structural changes were responsible for the increase of antioxidant
352
activity of α-LA.
353
Peptides possessing antioxidant activity are generally small in size, with
354
molecular weight not exceeding 3 kDa34 and some amino acid residues also possess
355
antioxidant activity, especially Cys, Trp, Tyr and Met35. Also, lien et al. reported that
356
Cys is an important element of the antioxidant system of the neonate36. The
357
hydrophobic amino acids Pro, His, Tyr, and Trp can serve as hydrogen donors33, thus
358
stabilizing free radicals thus stabilizing free radicals and accounting for protein’s
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inherent ability to act as an antioxidant. The structure of α-LA contains four Phe, four
360
Trp and four Tyr (Phe9, Phe31, Phe53, Phe80, Trp26, Trp60, Trp104, Trp118, Tyr18,
361
Tyr36, Tyr50 and Tyr103), which increase its potential to produce antioxidative
362
peptides. According to Leïla Sadat et al., the peptides 21VSLPEW26, 36YDTQA40,
363
101
364
scavenging capacity and contained at least one Tyr/Trp residue8. This finding can be
365
explained by the quenching of the Tyr-phenolic and Trp-indolic hydrogen (H+) by the
366
ABTS+ radicals to form more stable phenoxyl and indolyl radicals8, 37. The intrinsic
367
fluorescence spectra of all the samples decreased compared with that of N-LA in the
368
present work (Fig. 2), this result indicated that ultrasonic pretreatment combined with
369
glycation significantly affects the conformation of proteins changes around Tyr/Trp
370
residues38, 39. Moreover, during glycation a relevant variation in protein charge occurs
371
due to the involvement of the basic amino groups of proteins in Maillard-type
372
reaction40, this modification may induce the change of antioxidant activity. Therefore,
373
the antioxidant activity of α-LA increased after glycation with glucose.
INY103, 101INYW104 and 115LDQW118 possesses remarkable ABTS radical
In addition, the peptide 91CVKKILDKVGINY103 of all the samples contained the
374 375
antioxidant peptide 100INY103. After glycation, Cys91, Lys94 and Lys98 were
376
acetylated and glycated separately that led to the conformation of the peptide changes
377
around Cys91and Tyr103 residues, suggesting that the peptidic bond or structural
378
peptide conformation enhanced the antioxidant activity of the constitutive peptide and
379
amino acids37. However, four additional glycated peptides (104WLAHKALCSEKL115,
380
105
LAHKALCSEKLDQWL119, 105LAHKALCSEKLDQWL119 and
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381
111
382
was glycated, the conformation of the peptide 104-115 around Cys111 and Trp104
383
was changed, suggesting that the change of the structural peptide conformation
384
enhanced the antioxidant activity of the constitutive peptide. In the sample
385
ultrasonicated at 120 W/cm2 for 15 min, the glycated peptide 105-119 was identified
386
that includes antioxidant peptide 115LDQW118, the conformation of the peptide around
387
His107, Cys111, and Trp118 residues was similarly changed. Moreover, the peptides
388
105-119 and 111-123 were glycated, the same phenomenon around His107, Cys111,
389
Trp118 and Cys120 residues was observed to have actual change in sample
390
ultrasonicated at 150 W/cm2 for 15 min, and had finally improved antioxidant activity
391
of α-LA. The antioxidant activity of the increasing ultrasonic power levels was
392
observed in the order 150 W/cm2 > 120 W/cm2 > 90 W/cm2 > 0 W/cm2. Also, the DSP
393
value showed ultrasonic pretreatment promoted the glycation extent (Fig. 7).
394
Therefore, ultrasonic pretreatment increased the antioxidant activity of α-LA by
395
altering the conformation of α-LA and improving its glycation extent.
396
CSEKLDQWLCEKL123) were found after ultrasonic pretreatment. When Lys108
In summary, the results of the experiment demonstrated that ultrasonication
397
combined with glycation significantly improved the glycation and antioxidant
398
activities of α-LA. This finding was attributed to the covalent binding of glucose to
399
α-LA and to the succeeding structural changes of α-LA around some amino acid
400
residues. Moreover, ultrasonic pretreatment promoted the increase of antioxidant
401
activities by improving glycation. The result was reflected by the reduction in the
402
intrinsic fluorescence emissions and increase in the glycation sites and DSP value. It
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also demonstrated that high-resolution mass spectrometry is a powerful tool for
404
analyzing the protein modifications at a molecular level, thereby enabling the
405
exploration of the mechanism of improved antioxidant activity in food proteins during
406
food processing. Therefore, ultrasonication combined with glycation was revealed as
407
a good technology for improved antioxidant activity of proteins.
408
Acknowledgements
409
This work was supported by Chinese National Natural Science Foundation (No.
410
31360374), the earmarked fund for China Agriculture Research System (CARS-45),
411
Chinese National Natural Science Foundation (No. 31460395), and Excellent Youth
412
Foundation of Jiangxi Province (20162BCB23017).
413
References
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β-lactoglobulin and α-lactalbumin—Technological implications for processing. Int.
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(3) Meng, X.; Li, X.; Wang, X.; Gao, J.; Yang, H.; Chen, H., Potential allergenicity
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(9) Saricay, Y.; Wierenga, P. A.; De Vries, R., Limited changes in physical and
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emulsifying
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antigenicity of α-lactalbumin and β-lactoglobulin in whey protein conjugated with
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α-lactalbumin. Food Biophys. 2016, 11(1), 108-115.
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Enhancement of heat transfer by ultrasound: review and recent advances. Inter. J.
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(20) Zhang, Q.; Tu, Z.; Wang, H.; Huang, X.; Shi, Y.; Sha, X.; Xiao, H., Improved
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glycation after ultrasonic pretreatment revealed by high-performance liquid
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(21) Yang, W. H.; Tu, Z. C.; Wang, H.; Li, X.; Tian, M., High-intensity ultrasound
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enhances the immunoglobulin (Ig) G and IgE binding of ovalbumin. J. Sci. Food
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(22) Chandrapala, J.; Zisu, B.; Kentish, S.; Ashokkumar, M., The effects of
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high-intensity ultrasound on the structural and functional properties of α-Lactalbumin,
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β-Lactoglobulin and their mixtures. Food Res. Int. 2012, 48(2), 940-943.
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(23) Zhang, M.; Zheng, J.; Ge, K.; Zhang, H.; Fang, B.; Jiang, L.; Guo, H.; Ding, Q.;
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Ren, F., Glycation of α-lactalbumin with different size saccharides: Effect on protein
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Pang, J.; Yang, P., The reduction in the IgE-binding ability of β-Lactoglobulin by
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6179-6187.
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antioxidative activity of glycated α-lactalbumin with a rare sugar, D-allose, by
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(34) Kamau, S. M.; Cheison, S. C.; Chen, W.; Liu, X. M.; Lu, R. R.,
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(39) Li, C.; Xue, H.; Chen, Z.; Ding, Q.; Wang, X., Comparative studies on the
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(40) Harsha, P. S.; Lavelli, V.; Scarafoni, A., Protective ability of phenolics from
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albumin induced by glycation. Food Chem. 2014, 156, 220-226.
531
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Figure captions Fig. 1: Size exclusion chromatography (SEC) for analyses N-LA, N-LA-Glc, LAGlc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150. Fig. 2: The intrinsic fluorescence spectra of N-LA, N-LA-Glc, LA-Glc-60, LA-Glc90, LA-Glc-120 and LA-Glc-150. Fig. 3: The ABTS radical-scavenging activity (%) of N-LA, N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150. Different letters on the top of the bars denote significant difference (p < 0.05). Fig. 4: Mass spectra for the unglycated peptides of LA-Glc-150. (A) peptide 61-71 at m/z 434.51073+, (B) peptide 91-102 at m/z 462.92253+, (C) peptide 104-115 at m/z +
+
485.93093 , (D) peptide 105-119 at m/z 604.65383 , (E) peptide 111-123 at m/z 570.29263+. The determined peptides are labelled by residue numbers. The m/z differences between glycated and unglycated peptides are indicated above the arrows. Fig. 5: The ETD MS/MS spectra of the glycated peptides. (A) the glycated peptide C(carbamidomethyl)KDDQNPHSSN71 with m/z of 488.52813+, (B)
61
C(carbamidomethyl)VKKILDKVGIN102 with m/z of 570.97853+, (C) the glycated
91
peptide 91C(carbamidomethyl)VKKILDKVGINY103 with m/z of 428.73784+, (D) the glycated peptide 104WLAHKALC(carbamidomethyl)SEKL115 with m/z of 539.94843+, (E) the glycated peptide 105LAHKALC(carbamidomethyl)SEKLDQWL119 with m/z of 658.67163+. (F) the glycated peptide 111
C(carbamidomethyl)SEKLDQWLC(carbamidomethyl)EKL123 with m/z of
624.29033+. The sequence of per peptide is depicted on the top of the spectrum. The
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identified glycated sites are indicated by a line with glucose. The c and z ions are shown by the numbers and lines. Fig. 6: Ribbon diagram of the glycated α-LA (PDB 1F6S). The glycation sites are colored as follows: grey, framework of α-LA; red, glycation sites of the native α-LA; green, additional glycation sites of the α-LA after ultrasonication. Fig. 7: The average degree of substitution per peptide molecule (DSP) value of glycated sites of N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150.
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Fig. 1: Size exclusion chromatography (SEC) for analyses N-LA, N-LA-Glc, LAGlc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150.
N-LA N-LA-Glc LA-Glc-60 LA-Glc-90 LA-Glc-120 LA-Glc-150
100
Relative absorbance
80
60
40
20
0 15
16
17
18
19
20
21
22
Elution time (min)
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24
25
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Fig. 2: The intrinsic fluorescence spectra of N-LA, N-LA-Glc, LA-Glc-60, LA-Glc90, LA-Glc-120 and LA-Glc-150.
N-LA N-LA-Glc LA-Glc-60 LA-Glc-90 LA-Glc-120 LA-Glc-150
600
Fluorescence intensity
500
400
300
200
100
0 300
320
340
360
Wavelength (nm)
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400
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Journal of Agricultural and Food Chemistry
Fig. 3: The ABTS radical-scavenging activity (%) of N-LA, N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150. Different letters on the top of the bars denote significant difference (p < 0.05).
0.7
e
d 0.6
c
bc
b
0.5 0.4
a
0.3 0.2 0.1
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-1 50 lc G
LA -
LA -
G
lc
-1 20
-9 0
-6 0 lc
lc LA -G
-L N
LA -G
AG lc
-L A
0.0
N
ABTS radical-scavenging activity (%)
0.8
Journal of Agricultural and Food Chemistry
Page 30 of 36
Fig. 4: Mass spectra for the unglycated peptides of LA-Glc-150. (A) peptide 61-71 at m/z 434.51073+, (B) peptide 91-102 at m/z 462.92253+, (C) peptide 104-115 at m/z 485.93093+, (D) peptide 105-119 at m/z 604.65383+, (E) peptide 111-123 at m/z 570.29263+. The determined peptides are labelled by residue numbers. The m/z differences between glycated and unglycated peptides are indicated above the arrows.
AA (61-71)
A
100
80
Relative abundance
80
60
+3 434.5107
40
+3 m/z=54.0174 488.5281
20
80
60
40
AA (104-115)
C
+3 462.9225 m/z=54.0381
+3 570.9785
+3 516.9606
20
60
+3 485.9309
40
20
m/z=54.0175
+3 539.9484
m/z=54.0179 410
420
430
440
450
460
470
480
490
500
0 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
mass (m/z)
100
AA (105-119)
D
100
80
460
470
480
490
+3 604.6538
40
20
+3 658.6716
AA (111-123)
E
600
610
620
+3 570.2926
20
m/z=54.0178
590
60
40
630
640
650
660
670
680
0 550
500
510
mass (m/z)
80
60
0 580
0 450
mass (m/z)
Relative abundance
0 400
Relative abundance
Relative abundance
100
AA (91-102)
B
Relative abundance
100
+3 624.2903 m/z=53.9977
560
570
580
mass (m/z)
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590
600
610
mass (m/z)
620
630
640
650
520
530
540
550
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Fig.5: The ETD MS/MS spectra of the glycated peptides. (A) the peptide C(carbamidomethyl)KDDQNPHSSN71 with m/z of 488.52813+, (B) the peptide
61
C(carbamidomethyl)VKKILDKVGIN102 with m/z of 570.97853+, (C) the peptide
91
91
C(carbamidomethyl)VKKILDKVGINY103 with m/z of 428.73784+, (D) the peptide WLAHKALC(carbamidomethyl)SEKL115 with m/z of 539.94843+, (E) the peptide
104
105
+
LAHKALC(carbamidomethyl)SEKLDQWL119 with m/z of 658.67163 . (F) the
peptide 111C(carbamidomethyl)SEKLDQWLC(carbamidomethyl)EKL123 with m/z of 624.29033+. The sequence of per peptide is depicted on the top of the spectrum. The identified glycated sites are indicated by a line with glucose. The c and z ions are shown by the numbers and lines.
AA (61-71)
100
2 3 4 5 6 7 8 9 10 11
C KD D QN P H SS N 11 9 8 7 6 5 4 3 2
2 3 4 5 6 7 8 9 10 11 12
C VK K I L D KV G I N 12 1110 8 7 6
Glc
43 2
z
Glc
1326.7092,c81307.7015,z9 1425.7896,c9 1435.8795,z10 1482.8268,c10 1535.8318,z11+1 1595.8988,c11
6
1036.6016,c7 1017.6049,z8
40
20
0
676.3699,z5 695.5075,c4 791.4779,z6 809.5352,c5+1 921.6217,c 904.4004,z7
60
405.3303,c3
Relative abundance
1174.4836,c8
AA (91-102)
c
80
1261.4570,c9 1288.5302,z10+1 1348.6303,c10
997.2970,z9
882.2476,z8
B
z
Glc
1037.5393,c7
20
699.3088,c4+1 767.3072,z7
40
428.0748,z4 468.3496,c2
60
177.9824,c1
Relative abundance
80
826.2981,c5
c
277.2514,c2
A 583.2476,c3 639.2216,z6
100
0 200
400
600
800
1000
1200
1400
1600
200
400
mass (m/z)
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600
800
1000
1200
mass (m/z)
1400
1600
1800
Relative abundance 40
20
200 400
80
60
600 800 1600
15 14 13 12 1110 9 8 7
Relative abundance
1433.9200,c11
1180.6198,z91164.6140,c8 1263.7394,c9 1309.6865,z10+1 1320.7831,c10
20
L A H K A L CS E K L D QW L
5 4 3 2
0 1000 1200 1400 1600 1800 2000 z
1800 200
100
20
200 400
mass (m/z)
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80
Glc
80
600
mass (m/z) 600 800
F
60
40
800
c
12 11 10 9
1000
2 3 4 5 6 7 8 9 10 1112 13 14 15
mass (m/z)
1200
1504.7905,c11+1
1094.6017,z8 1159.6459,c8 1232.6746.z9+1 1246.6782,c9 1303.7139.z10+1 1375.7249,c10 1416.7567,z11+1
999.6705,c7
733.5787,z6 804.5662,z7 815.5378,c 5 886.5884,c6
622.5347,z5+1
460.5051,z4 525.5196,c4
373.4115,z3
244.2994,z2
z
1756.7581,c12
c 1400
40
c
1466.6117,c11 1479.6781,z10+1 1608.7542,z11+1
0
D
1350.6132,z9
1200
60
1122.5525,z7
717.5822,z112+ 774.4967,c122+
Glc
994.6263,z6 1064.4672,c8
1000
5 43 2
750.3962,c6
800 1067.5609,z8
759.5551,c6 838.4581,z +1 874.6055,c7 6 954.4203,z7
405.1248,c3
C VK K I L D KV G I N Y
808.3806,z5 878.6089,c7
E
13 12 11 10 9 8 7
100
406.3001,z2
600
AA (91-103)
522.3035,c4 535.3342,z 635.4223,c5 3
400
2 3 4 5 6 7 8 9 10 11 12 13
Relative abundance
200
c
1437.5723,z10 1509.6375,z11+1 1545.7932,c12 1636.6967,z12 1675.8352,c13+1 1774.1211,z13+1 1845.8638,z14+1
20
811.5647,c7 898.5483,c8 930.7515,c14+12+ 1028.6371,c9 1077.5889,z7 1164.6593,z8 1316.6409,c10+1
60 277.1412,c2
80
650.7637,c6+1
100
C
467.2661,c4
40
393.2061,z3 450.2657,z4 533.4204,c4 549.2681,z5 645.6258,c5+1
100
339.1884,c3
Relative abundance
Journal of Agricultural and Food Chemistry Page 32 of 36
2 3 4 5 6 7 8 9 10 11 12
AA (104-115)
W L A H K A L C S EK L Glc
7 6 54 3 2
1400
13 12 1110 9 8 7 6 5 4 3
C S E K L D QW LC E K L
z
0
mass (m/z)
1600
AA (105-119)
2 3 4 5 6 7 8 9 10 1112 13
AA (111-123)
Glc
z
0 1000 1200 1400 1600 1800 2000
Page 33 of 36
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 native α-LA; green, additional glycation sites of the α-LA after ultrasonication.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Fig. 7: The average degree of substitution per peptide molecule (DSP) value of glycated sites of N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150.
N-LA-Glc LA-Glc-60 LA-Glc-90 LA-Glc-120 LA-Glc-150
1.0
0.8
DSP
0.6
0.4
0.2
0.0 61-71
91-102
104-115
105-119
peptide
ACS Paragon Plus Environment
111-123
Page 34 of 36
Page 35 of 36
Journal of Agricultural and Food Chemistry
Table 1. Summary of the glycated peptides in the N-LA-Glc, LA-Glc-60, LA-Glc-90, LA-Glc-120 and LA-Glc-150. Peptide
Sequence
488.52833+
1.09
(W)C*KDDQNPHSSN(I)
K62
91-102
570.9784
3+
0.70
(M)C*VKKILDKVGIN(Y)
K94,K98
91-103
428.73804+
0.53
(M)C*VKKILDKVGINY(W)
K98
61-71
488.52833+
0.07
(W)C*KDDQNPHSSN(I)
K62
91-102
570.9768
3+
2.69
(M)C*VKKILDKVGIN(Y)
K94,K98
91-103
428.73774+
-5.96
(M)C*VKKILDKVGINY(W)
K98
104-115
539.94873+
1.79
(Y)WLAHKALC*SEKL(D)
K108
61-71
488.52803+
0.89
(W)C*KDDQNPHSSN(I)
K62
91-102
570.97823+
2.81
(M)C*VKKILDKVGIN(Y)
K94,K98
91-103
428.73764+
-6.31
(M)C*VKKILDKVGINY(W)
K98
3+
-0.31
(Y)WLAHKALC*SEKL(D)
K108
61-71
488.52813+
0.55
(W)C*KDDQNPHSSN(I)
K62
91-102
570.97873+
3.04
(M)C*VKKILDKVGIN(Y)
K94,K98
91-103
571.3154
3+
1.34
(M)C*VKKILDKVGINY(W)
K98
104-115
539.94873+
1.36
(Y)WLAHKALC*SEKL(D)
K108
105-119
494.25304+
-3.50
(W)LAHKALC*SEKLDQWL(C)
K114
61-71
488.52813+
0.96
(W)C*KDDQNPHSSN(I)
K62
91-102
570.97853+
1.11
(M)C*VKKILDKVGIN(Y)
K94,K98
91-103
428.73794+
-5.67
(M)C*VKKILDKVGINY(W)
K98
104-115
539.9484
3+
-0.12
(Y)WLAHKALC*SEKL(D)
K108
105-119
658.67163+
0.66
(W)LAHKALC*SEKLDQWL(C)
K114
111-123
624.29043+
0.37
(L)C*SEKLDQWLC*EKL
K122
location
(m/z)
N-LA-G
61-71
LA-Glc-90
104-115 LA-Glc-120
LA-Glc-150
Glycated Δppm
Sample
LA-Glc-60
a
Glycated peptide
539.9499
C* refers to carbamidomethyl.
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a
site
Journal of Agricultural and Food Chemistry
Page 36 of 36
Graphical abstract
Native α-LA Ultrasonic pretreatment Dry heating glycation with glucose
Dry heating glycation with glucose Glucose
Glycation extent
Antioxidant activity
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