Liquid Chromatography High-Resolution Mass Spectrometry Identifies

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Liquid chromatography high-resolution mass spectrometry identifies the glycation sites of bovine serum albumin induced by D-ribose with ultrasonic treatment Nanhai Zhang, Zongcai Tu, Hui Wang, Guangxian Liu, Zhenxing Wang, Tao Huang, Xu Qin, Xing Xie, and A'mei Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04578 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on January 1, 2018

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Journal of Agricultural and Food Chemistry

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Liquid chromatography high-resolution mass spectrometry identifies the

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glycation sites of bovine serum albumin induced by D-ribose with ultrasonic

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treatment

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Nanhai Zhang,a Zongcai Tu,a,b,* Hui Wang,a,* Guangxian Liu,b,c Zhenxing Wang,b Tao

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Huang,a Xu Qin,a Xing Xie,a A’mei Wanga

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a

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Nanchang, Jiangxi 330047, China

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b

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China

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c

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

College of Life Sciences, Jiangxi Normal University, Nanchang, Jiangxi 330022,

Jiangxi Academy of Agricultural Sciences, Nanchang, Jiangxi 330200, China

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* Corresponding author:

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Prof. Zong-cai Tu,

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99 Ziyang Road, Nanchang, Jiangxi, China

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Fax: +86-791-8812-1868

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Phone number: +86-791-8812-1868

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E-mail: [email protected] (Zongcai Tu)

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Dr. Hui Wang,

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235 East Nanjing Road, Nanchang, Jiangxi, China

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Fax: +86-791-8830-5938

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Phone number: +86-791-8830-5938

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E-mail: [email protected] (Hui Wang)

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ACS Paragon Plus Environment


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ABBREVIATIONS USED

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MR, Maillard reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel

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electrophoresis;

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spectrometry; HCD, higher-energy collisional dissociation; ETD, electron transfer

28

dissociation; BSA, bovine serum albumin; DHPM, dynamic high pressure

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microfluidisation; DTT, dithiothreitol; MS, mass spectrometry; MS/MS, tandem mass

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spectrometry; UV, ultraviolet; DSP, the average degree of substitution per peptide

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molecule; Trp, tryptophan; Tyr, tyrosine; HPLC, high performance liquid

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chromatography; UA, urea; IAA, indole-3-acetic acid; Phe, phenylalanine

LCHR-MS,

liquid

chromatography

2


high-resolution

mass

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ABSTRACT

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Ultrasonication is an emerging technology applied in food processing and biological

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experimental pretreatments. Cavitation phenomena induced during ultrasonic

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treatment can generate localized high temperature and pressure, which can result in

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glycation reaction between protein and reducing sugars. In this study, the mixture of

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bovine serum albumin (BSA) and D-ribose was treated under 600 W for different

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times. Interestingly, a large amount of carbonized black materials appeared after

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ultrasonication, while the UV absorbance and intrinsic fluorescence spectra reflecting

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conformational changes were not obvious. Only 12 sites (11 lysines and 1 arginine) of

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the BSA with ribose under ultrasonic treatment for 35 min were identified through

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liquid chromatography high-resolution mass spectrometry (LCHR-MS). K547, K548,

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R359/R360, and K587 were the most reactive glycated sites, with the average degree

45

of substitution per peptide molecule (DSP) value ranging from 15% to 35%. The

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glycated modification was distributed not only in domain III, but also in domain I and II.

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The glycated modification could occur during ultrasonic treatment, thereby influencing

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the properties of biomacromolecule after extraction.

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Keywords: BSA, Ultrasonication, Glycation, LCHR-MS, DSP

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INTRODUCTION

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Ultrasound is a high-frequency sound wave that exceeds the upper limit of human

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hearing frequency range (~20 kHz). When powerful ultrasound is interacting with

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liquids, its nucleus grows; then the formed gas bubble implosively collapses thereby

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inducing acoustic cavitation phenomena as the main effect of ultrasound.1 Cavitation

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generates immense pressure, high temperature and local turbulence that can alter

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physicochemical and functional properties of materials.2 Thus, ultrasonic treatment is

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wildly applied in many processing industries (such as chemical, pharmaceutical and

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cosmetic industries) and experiment pretreatment.3-4 Ultrasound, has been particularly

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applied in bio-macromolecule processing for the extraction of proteins and for

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improving their extracting rate,5-6 breaking the cell walls to release DNA rapidly from

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the microorganisms,7 and for the reducing of the viscosity and the relative molecular

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weight of the kappa carrageenan in order to promote their applications.8

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The large energy concentration generated from the gas bubbles collapse transforms

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the kinetic energy of the liquid motion to thermal energy, thereby generating highly

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localized temperature of approximately 5000 K and pressures exceeding several

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thousand bars.9 Ultrasonication can lead to the chemical reaction of protein and

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reducing sugars, such as Maillard reaction (MR), because it can provide enough

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energy and free amino groups for graft reaction,10 despite the well effect on extraction 4


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of proteins and nucleotides. MR can influence aroma, colour and nutritional value of

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food,11 and some studies have shown that dietary MR products might be correlated

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with health problems such as diabetes and chronic renal failure and could affect

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inflammatory markers.12-13 However, a few scholars realized this point and studied

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it.14 Therefore, the occurrence of glycation of protein and reducing sugars during

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ultrasonication and the mechanism of glycation during ultrasonication should be

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explored.

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Glycation is the first step of the MR, which occurs between amino group residues and

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the reducing sugars. Many experiments have revealed that the ultrasonic pretreatment

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of the mixture of protein and sugars can promote the formation and the

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physicochemical and functional properties of glycation products due to structural

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changes of protein by ultrasonication.14-15 Ultrasonic treatment can accelerate the

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glycation reaction of β-lactoglobulin with various sugars in aqueous model systems

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under neutral conditions, can improve antioxidant properties, and can induce minor

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changes in secondary and tertiary structures.16 Mu et al.10 employed ultrasonication to

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accelerate the graft reaction of soy protein isolate and gum acacia and to significantly

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increase the emulsifying properties. However, the mechanism of glycation during

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ultrasonication remains ambiguous.

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The extent of the glycation reaction has been recently assessed mostly by measuring

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the ultraviolet (UV) absorbance and browning intensity of the reaction products,17 free

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amino acid groups content of glycated proteins,10 ﬂuorescence intensity of glycated

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proteins,18 and the sodium dodecyl sulfate–polyacrylamide gel electrophoresis

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(SDS-PAGE) of the reaction products.19 All these methods can provide the

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microscopical changes in proteins but cannot find the initial glycated products，

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without reflecting the glycation site(s), and the glycation extent in every protein sites.

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In recent years, liquid chromatography high-resolution mass spectrometry

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(LCHR-MS), particularly with higher-energy collisional dissociation (HCD) and

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electron transfer dissociation (ETD), has been a powerful tool in enabling the

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unambiguous characterization of peptides with various modifications and evaluating

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the interaction between amino acid and reducing sugar.16 For example, Tu et al.20

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determined eight glycated sites from ovalbumin glycated with glucose under

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freeze-drying processing using LCHR-MS. Our previous study demonstrated that the

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glycation of the bovine serum albumin (BSA) with D-galactose was improved by

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ultrasonic pre-treatment and this was followed by LCHR-MS.21

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In the current study, the mixture of BSA (model protein) and D-ribose was treated

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under ultrasonication at 600 W for 0, 5, 10, 15, 20, 25, 30, and 35 min. Then, the

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SDS-PAGE, UV, and intrinsic emission fluorescence spectra of the mixture were

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explored. The glycated sites and the average degree of substitution per peptide

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molecule (DSP) were characterized by LCHR-MS combined with HCD

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fragmentation to probe the mechanism of glycation during ultrasonic treatment.

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MATERIALS AND METHODS

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Materials

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BSA (V9000933), D-(-)-ribose(V900389) and pepsin (P6887) were purchased from

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Sigma-Aldrich (St. Louis, MO, USA). Dithiothreitol (DTT) was purchased from

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Thermo Fisher Scientific Inc. (Waltham, MA, USA). All other chemical reagents were

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of analytical grade and high performance liquid chromatography (HPLC) grade.

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Ultrapure water used in the experiments was purified from a water purification system

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(Millipore, Billerica, MA, USA).

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Sample Preparation

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The mixture of BSA and ribose (at a mass ratio of 1:1) was dissolved in the Tris-HCl

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buffer (200 mM, pH 8.0) to obtain a final protein concentration of 10 mg/mL. The 7



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solution was split into eight aliquots. Sonication of the eight aliquots was carried out

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using an ultrasonic cell disruption instrument (JY98-IIIDN, Ningbo Xinzhi

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Instruments, Inc., Ningbo, China, 20kHz) with a 2.0 cm diameter probe. The sample

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vessel was kept in an ice bath, and the solution was treated under 600 W of power

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output for 0, 5, 10, 15, 20, 25, 30, and 35 min with a pulsation turned 1 and 3 s,

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respectively. Then the sample solution after ultrasonication was filtered with a

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Centricon centrifugal filter unit (10 kDa cutoffs, Millipore, Bedford, MA, USA) to

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remove the free ribose and salts. The centration of protein was adjusted to 1 mg/mL,

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and the resultant was stored at 4 °C for subsequent use. The mixture of BSA and

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ribose with ultrasonic treatment for 0, 5, 10, 15, 20, 25, 30, and 35 min was denoted

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as NU-BSAR, U-BSAR-5, U-BSAR-10, U-BSAR-15, U-BSAR-20, U-BSAR-25,

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U-BSAR-30 and U-BSAR-35, respectively.

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SDS-PAGE

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SDS-PAGE was performed using a vertical gel electrophoresis cell (Bio-Rad,

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Richmond, CA, USA) following a modified method of Laemmli.22 Stacking and

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separating gels at 5% and 12%, respectively, were prepared. Prior to electrophoresis,

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the samples were mixed with loading buffer. Then, the resultant was heated for 7 min.

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Ten microliters of the mixed solution were put on the gel with an initial voltage of

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80 V for 30 min and then at 100 V for 1.5 h. Thereafter, gels were stained with 0.05% 8


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Coomassie Brilliant Blue R-250 and destained in a mixture of 5% methanol and 7.5%

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acetic acid. Low molecular weight markers (14.4-97.4 kDa) were used for a molecular

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weight reference of samples.

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UV Absorption and Intrinsic Fluorescence Spectroscopy

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UV absorption spectra of samples were measured with a U-2910 spectrophotometer

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(Hitachi, Tokyo, Japan). UV absorption spectra were recorded from 260 nm to

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300 nm.18

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The intrinsic emission fluorescence spectra of the protein samples (1 mg/mL) were

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obtained with an F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The

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protein solutions were excited at 290 nm, and emission spectra were scanned from

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320 nm to 370 nm at a slit width of 5 nm for excitation and emission.23

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Sample Digestion

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Two hundred micrograms of U-BSAR-35 were added to a centrifuge tube containing

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an amount of DTT to obtain a final sample concentration of 100 mM. The sample was

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incubated in boiling water bath for 5 min and then cooled in an ice bath. Two hundred

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micrograms of urea (UA) buffer (8 M urea and 150 mM Tris-HCl buffer at pH 8.0) 9



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were added to the tube, and centrifuged at 14000 g for 15 min, and the filtrate was

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thrown away. One hundred microliters of indole-3-acetic acid (IAA) (50 mM IAA in

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UA buffer) was added to the above tube and oscillated at 600 rpm for 1 min. After 30

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min of incubation in the dark at room temperature, the mixture was centrifuged at

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14000 g for 10 min. One hundred microliters of UA buffer and HCl buffer (pH 2.2)

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were added to the above tube in order and the mixture was centrifuged. One hundred

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microliters of HCl buffer (pH 2.2).

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Forty microliters of pepsin solution (40 µg pepsin in 40 µl pH 2.2 HCl buffer) was

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added, and the mixture was stored in a 4 °C refrigerator for 10 min. Then, the mixture

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was centrifuged, and the filtrate was used to the analysis of mass spectrometry (MS).

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Analysis by HPLC-HCD-MS/MS

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The glycated sites and glycation extent were investigated following the method of Tu

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et al.20 The effluent was infused into an LTQ-Orbitrap Velos mass spectrometry

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(Thermo Fisher Scientiﬁc, Waltham, MA, USA) for tandem mass spectrometry

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(MS/MS) analysis to identify protein glycation though positive ion detection. A total

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of 20 fragment maps (MS/MS2 scans) reflecting the mass-to-charge ratio of peptides

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and their fragments were collected after each full scan. The ions detected in the 10


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precursor ion scanning were further subjected to HCD fragmentation to detect the

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fragment ions. Dynamic exclusion was enabled with an exclusion duration of 90 s.

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To further compare the glycation extent of each peptide, the DSP of BSA was

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calculated according to the following formulation21:

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DSP =

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Where I is the intensity of various glycated BSA peptides, and i is the number of ribose

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units attached to the peptide in each glycated form.

∑ in= 0 i × I (peptide + i × ribose) ∑ in= 0 I (peptide + i × ribose)

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RESULTS AND DISCUSSION

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SDS-PAGE

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As shown in Figure 1B, the carbonized black substances increased at the bottom of the

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centrifuge tubes with the prolonging of ultrasonic time. On the one hand, sugars could

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be caramelized, and then the carbonized black substances were produced due to the

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localized high temperature produced by ultrasonication. On the other hand, protein

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could be carbonized due to the cavitation effect that generates transient high

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temperature during ultrasonication. The ultrasonic treatment used in this experiment 11



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was equipped with a cooling system that could keep the sample at a low temperature,

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while protein could change owing to the localized hot spots produced by

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ultrasonication.

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SDS-PAGE profiles of the samples are shown in Figure 1A. Compared with NU-BSAR,

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no significant mobilities of bands between samples under ultrasonic treatment with the

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prolonging of time were found on the SDS-PAGE, suggesting that ultrasonication did

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not change the profile of BSA under our experiment conditions. However,

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Stanic-Vucinic et al.16 found that protein bands exhibit a minor shift after

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ultrasonication in the presence of some sugars, thereby resulting in dimer formation;

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therefore, high-intensity ultrasound can induce the glycation of β-lactoglobulin-ribose.

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In the previous study, the changed molecule weight of β-lactoglobulin could be

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observed in SDS-PAGE probably because of its small molecular weight. Nevertheless,

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the molecular weight of BSA was larger than that of β-lactoglobulin, which was

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responsible for the unobvious mobilities of bands. Therefore, LCHR-MS should be

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used to prove whether the glycation modification of protein occurred.

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UV Absorption and Fluorescence Spectra Analysis

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The UV absorption spectra of protein were primarily dependent on the absorption of 12


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light by side chain groups of chromophoric amino acids, such as tryptophan (Trp),

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tyrosine (Tyr), and phenylalanine (Phe). The absorption peaks of Trp and Tyr were at

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280 nm, while the absorption peak of Phe was at 257 nm.24 When conformation of a

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protein changed, the chromophores would be exposed to the surface of the protein,

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thereby increasing the intensity of absorption peak. As shown in Figure 2A, the

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maximum absorption peak was at 276 nm around 280 nm, demonstrating that the UV

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absorption spectrum of BSA was depended on Trp and Tyr residues. UV absorption

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intensity increased at different levels during ultrasonication from 5 min to 25 min.

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The increase in absorption suggested that Trp and Tyr residues were exposed on the

229

surface of the protein molecule, as a consequence of unfolding of the structure.

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Compared with NU-BSAR, U-BSAR-30 had a very slight decrease in the intensity of

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the maximum absorption peak. Protein refolding could occur at different levels during

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ultrasonication from 5 min to 30 min, which was attributed to the localized high

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temperature produced by ultrasonication.25 U-BSAR-35 had the highest intensity due

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to the largest structural changes. In general, the UV absorption intensity under

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ultrasonication had insignificant differences.

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The intrinsic fluorescence spectra of protein were attributed to the polarity of the

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environment of the Trp residues or their specific interactions. The intensity of

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fluorescence would decrease when the chromophores were exposed to a solvent.26 As

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presented in Figure 2B, NU-BSAR exhibited a fluorescence emission maximum at

241

342 nm, when excited at 290 nm. Notably, compared with NU-BSAR, the ultrasonic

242

treatment increased fluorescence intensity from 5 min to 20 min, whereas decreased

243

the intensity from 25 min to 35 min. Fluorescence intensity declined as the treatment

244

time increased, and the wavelength of fluorescence emission peak was not shifted.

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The increase in intensity indicated the gradual exposure of Trp toward hydrophilic

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surroundings. As a result, conformational changes in protein occurred in the vicinity

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of the Trp residues because of the thermal denaturation of protein by ultrasound.15

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Under ultrasonic treatment for 25−35 min, the decline in fluorescence intensity was

249

due to the quenching of fluorescence, indicating that Trp could contact with polar

250

solvent.18 Therefore, the fluorescence spectrum analysis indicated that disruption of

251

the tertiary structure of BSA could occur insignificantly under ultrasonic treatment,

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thereby exposing the chromophores to solvent.

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254

The insignificant differences in UV absorption spectra and the slight changes in

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intrinsic fluorescence emission spectra indicating that the reaction was at the early

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stage of MR could be due to the steric hindrance effects on the 3D structure of protein

257

in ribose solvent.27 Some functional properties of food proteins such as

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radical-scavenging activity, reducing power, etc. could be improved with this

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structural change,16 thereby possibly affecting the quality of food proteins.

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Peptide Mapping and Glycation Identification

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U-BSAR-35 was used for MS measurement because of its significant change in UV

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and intrinsic fluorescence spectra, which could result in the highest glycation extent.

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Prior to understanding the influence of ultrasonication treatment on the glycation

265

reaction of BSA, the complete peptide map of the glycated sites of BSA with and

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without ultrasonication treatment should be first obtained. We performed pepsin

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digestion followed by LCHR-MS/MS. Pepsin was selected as the non-specific

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protease because of its excellent property in cleaving peptide bonds between

269

hydrophobic residues. Accordingly, small peptides beneficial to subsequent MS

270

analysis could be generated and the difference in protein structure in the digestion

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process could be decreased.28 Then, the peptic peptides identified and determined by

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LCHR-MS/MS were listed in Table S1 (provided in the Supporting Information).

273

274

The structure of the glycated BSA could be analyzed by identifying the glycation sites.

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To identify the glycated amino acid residues, we performed MS/MS with HCD after

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pepsin digestion. The MS spectra of the representative glycated peptides of the

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BSA-ribose under ultrasonic treatment were shown in Figure 3. Then, the glycation

278

peptides could be determined from the mass shift. Theoretically, if a peptide was

15



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glycated by one molecule of ribose via the early stage of MR, accompanied by loss of

280

a water molecules,11 then the corresponding m/z peaks with charges of 1, 2, and 3

281

would display a mass increase of 132 Da with m/z changes of 132, 66, and 44,

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respectively.16 For dual-glycated peptides, the mass increase would be 264.0845 Da.

283

For example, the m/z peaks of the unglycated peptides 355-364 and 570-580 (as

284

shown in Figure 3B and 3D) were 467.21813+ and 433.55173+, while the

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corresponding m/z peaks of glycated peptides had an increase in m/z of 44.0140.

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Similarly, the ion peaks with m/z of 619.79622+ and 535.76152+ were identified as the

287

unglycated peptides 179-188 and 419-427 (as shown in Figure 3A and 3C), while the

288

new peaks with the m/z values at 685.81692+ and 601.78232+ had an increase in m/z of

289

shift 66.0211. The m/z differences with the increase in mass of 132 Da indicated that all

290

these peptides were modified by one molecule of ribose under ultrasonic treatment.

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Figure 3A shows that the m/z peak of the unglycated peptide 546-552 was 401.26292+.

292

New peaks with the m/z values at 467.28432+ and 533.78182+ with a mass shift of 132

293

and 264 Da, respectively, were found. Therefore, the peptide attached two molecules of

294

ribose.

295

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The possible and preferable glycation groups were the α-amino group of N-terminus,

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the ε-amino groups of lysine residues and the guanidine groups of arginine residues

298

during the MR process.29 In this paper, HCD MS/MS was performed to locate the exact

16


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glycation sites, especially for peptides with multiple glycation sites. For example,

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Figure 4B and 4C show the HCD MS/MS spectrum of the dual-glycated peptide

301

546-522 with the sequence of IKKQTAL at m/z 467.28432+ and 533.78182+.

302

Considering the compete matching of b and y fragment ions and according to Figure 4A,

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K547 and K548 were the glycated sites. Similarly, Figure S1A shows the HCD MS/MS

304

spectrum of the single glycated peptide 179-188 at m/z 685.81692+. A series of detected

305

b and y fragment ions fully matched with the fragmentation of the peptide

306

YYANK(gly)YNGVF, thereby confirming that the glycation site occurred at K183.

307

Similarly, K420 was identified as the glycation site of the glycated peptide 419-427

308

(EKLGEYGFQ) at m/z 601.78232+ (Figure S1B).

309

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Figure S2 shows the peptide 243-251 contained two possible glycation sites at K245

311

and K248 and fragment ions that fully matched with the fragmentation of the peptide.

312

However, we could not exactly identify the glycation site of this peptide due to the

313

limited MS/MS sensitivity. The same situation occurred at the peptide 355-364

314

(YEYSRRHPEY), which contained two possible glycation sites at R335 and R336. The

315

other glycated sites were identified from the fragmentation of the corresponding

316

glycation peptides (Table 1). Only 12 sites of U-BSAR-35 were identified, including 11

317

lysine residues and 1 arginine residue. The ε-amino groups of the lysine residues were

318

the major reactive groups in the MR during ultrasonic treatment. This result was in

17



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accordance with the previous studies, wherein glycated sites predominantly located at

320

lysine but not at arginine.20-21,

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U-BSAR-35, which was also observed in glycated BSA.21 Therefore, this finding

322

shown that the glycation of BSA occurred under ultrasonic treatment for 35 min at the

323

presence of ribose.

30

Moreover, arginine residue was modified in

324

325

Glycation Extent and Structural Changes of BSA During Ultrasonic Treatment

326

The DSP of each glycated peptide should be calculated to evaluate the degree of the

327

glycation (as shown in Table 1) and thus understand the relative glycation reactivity of

328

peptides during ultrasonic treatment. With regard to DSP values, K547, K548,

329

R359/R258, and K587 with a DSP of up to 15% were the most reactive sites to

330

glycation with ribose in BSA. However, most of DSP values ranged from 1% to 7%

331

for other peptides. Glycated peptides were distributed in several parts of the protein,

332

where structural perturbation occurred (Figure 5). This finding suggested that

333

ultrasonic treatment could induce a structural change in protein,21 the exposing a large

334

number of reactive sites of the protein,31 and thereby obtaining high probability of

335

glycation.16

336

337

The peptide 1-18 is a signal peptide and the following peptide 19-24 is a basic 18


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propeptide, which are cleaved during protein maturation or activation.32 Therefore, the

339

mature BSA sequence included 583 residues. The glycation sites in Table 1 were 24

340

times than the marking glycation sites in Figure 5. The glycation sites identified above

341

demonstrates that the protein structure was perturbed by ultrasonication. BSA was

342

composed of three domains, each of which was formed by six helices, including

343

domain I comprising residues 1-184, domain II comprising residues 185-377, and

344

domain III comprising residues 378-583.33 As shown in Figure 5A, the glycation sites

345

covered all three domains, including 3, 4, 5 glycation sites in domain I, II, and III.

346

347

In the tertiary structure of BSA (Figure 5A), the glycated sites were mainly located at

348

the α-helix and few located at random coil and β-turn. K41 and K396 were situated on

349

the surface of the spherical protein, where the glycation occurred under

350

ultrasonication. K523 and K524 had a very high reactivity for glycation because its

351

neighboring amino acid was Ile, which could play a role in increasing lysine reactivity

352

and promoting glycation.34 K563, which was located at the outer surface of the

353

hydrophilic area in the tertiary structure, also had a high extent of glycation. The

354

reason was due to its several surrounding hydrophilic amino acids, including Glu548,

355

Phe550, Val551, Ala552, Phe553, Val554, Asp555, Lys(K)556, Ala559, Ala560,

356

Asp561, Asp562, Lys(K)563, Glu564, Ala565, Phe567, Ala568, Val569, Glu570,

357

Pro572, Lys573, and Val575 (as shown in Figure 5B).20 Therefore, ribose could be

19



358

easily closed to K563 and K556 with the surrounding hydrophilic amino acids.

359

Similarly, K221/K224 was a glycated site because of its neighboring Phe and several

360

hydrophilic amino acids. R335/R336 exhibited a very high extent of glycation. This

361

result could be attributed to the opening of BSA structure, thereby allowing ribose to

362

be close to R335/R336. H337, which was the catalytic basic residue, might help

363

promote glycation.29 With the structure of BSA unfolded, K294 and K350 distributed

364

in domain II were also glycated with ribose. In addition, K106 and K159 were

365

situated at the inner part of the hydrophilic pocket of domain I such that several

366

changes occurred in domain I. Given the partial unfolding of protein, Trp probe

367

(Trp134 and Trp213) might rapidly and largely exposed to the solvent, thereby

368

changing the fluorescence spectra.35

369

370

The glycation reaction of BSA occurred not only in domain III but also in domains I

371

and II during ultrasonic treatment at the presence of ribose owing to the structural

372

changes of BSA. This result conforms with a previous study, wherein they reported

373

that the increasing glycation sites induced by ultrasonic pretreatment are also

374

distributed throughout the three domains.21 However, the number of glycated sites in

375

the present study (Table 1) was fewer than the number reported by Zhang et al.21, and

376

the current glycation sites were slightly different from those by Zhang et al.21 The

377

reason could be that we applied ultrasonication on protein–saccharide, whereas Zhang

20


Page 20 of 34

Page 21 of 34


378

et al.21 applied ultrasonication only on protein combined with glycation. Furthermore,

379

the treatment time in the present study was longer than that by Zhang et al.21 A

380

previous study demonstrated that the glycation sites of BSA induced by dynamic high

381

pressure microfluidisation (DHPM) pretreatment are mainly in domains II and III not

382

in domain I. A similar result was reported on BSA denaturation induced by

383

guanidinium hydrochloride.36-37 DHPM induces high energy in a short time whereas

384

ultrasonication produces high energy in a long time, thereby resulting in the difference

385

between the current and previous results.

386

387

In the present study, SDS-PAGE, UV, and intrinsic fluorescence spectra reflecting

388

conformational changes had no obvious differences. Twelve glycated sites (11 lysines

389

and 1 arginine) were identified using LCHR-MS/MS, and the DSP of the most

390

reactive ribose glycation sites in BSA were up to 15%. The glycated modification was

391

distributed not only in domain III, but also in domain I and II. This result was

392

attributed to the structural changes of BSA under ultrasonic treatment for 35 min at

393

the presence of ribose and covalent binding of ribose to BSA. On the one hand,

394

ultrasonic treatment could be applicable to facilitate physical and functional properties

395

of food proteins via changing its structure and improving glycation. On the other hand,

396

this result could instruct the extraction of biomolecules such as proteins and

397

nucleotides through controlling the power or time of ultrasonic treatment to protect

21



398

the biomolecules’ quality.

399

400

ACKNOWLEDGEMENTS

401

This work was supported by the National Natural Science Foundation of China

402

(NSFC) (No. 21706111), the earmarked fund for China Agriculture Research System

403

(CARS-45), and Jiangxi province science and technology support program

404

(20161BBF60021).

405

406

SUPPORTING INFORMATION

407

Table S1, Figure S1 and Figure S2

408

409

REFERENCES

410 411 412 413 414 415 416 417 418 419 420

(1) Rooze, J.; Rebrov, E. V.; Schouten, J. C.; Keurentjes, J. T., Dissolved gas and ultrasonic cavitation--a review. Ultrason. Sonochem. 2013, 20 (1), 1-11. (2) Awad, T. S.; Moharram, H. A.; Shaltout, O. E.; Asker, D.; Youssef, M. M., Applications of ultrasound in analysis, processing and quality control of food: A review. Food Res. Int. 2012, 48 (2), 410-427. (3) Bhattacharya, D. M.; Pratap, U. R.; Wankhade, A. V.; Zodape, S. P., Volumetric and ultrasonic approach in the investigation of critical micellar phenomenon of amphiphilic drugs in aqueous solutions at different temperatures. J. Mol. Liq. 2016, 214, 117-127. (4) Mahmoodi, N. O.; Mohammadi Zeydi, M.; Biazar, E., Ultrasound-promoted one-pot four-component synthesis of novel biologically active 3-aryl-2,4-dithioxo-1,3,5-triazepane-6,7-dione and their toxicity investigation. J. 22


Page 22 of 34

Page 23 of 34


421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462

Sulfur Chem. 2016, 1-9. (5) Kadam, S. U.; Tiwari, B. K.; Álvarez, C.; O'Donnell, C. P., Ultrasound applications for the extraction, identification and delivery of food proteins and bioactive peptides. Trends Food Sci. Technol. 2015, 46 (1), 60-67. (6) Vilkhu, K.; Mawson, R.; Simons, L.; Bates, D., Applications and opportunities for ultrasound assisted extraction in the food industry — A review. Innov. Food Sci. Emerg. Technol. 2008, 9 (2), 161-169. (7) Hohnadel, M.; Felden, L.; Fijuljanin, D.; Jouette, S.; Chollet, R., A new ultrasonic high-throughput instrument for rapid DNA release from microorganisms. J. Microbiol. Methods 2014, 99, 71-80. (8) Azizi, R.; Farahnaky, A., Ultrasound assisted-viscosifying of kappa carrageenan without heating. Food Hydrocolloids 2016, 61, 85-91. (9) Suslick, K. S.; Didenko, Y.; Fang, M. M.; Hyeon, T.; Kolbeck, K. J.; McNamara, W. B.; Mdleleni, M. M.; Wong, M., Acoustic cavitation and its chemical consequences. Phil. Trans. R. Soc. Lond. A 1999, 357 (1751), 335-353. (10) Mu, L.; Zhao, M.; Yang, B.; Zhao, H.; Cui, C.; Zhao, Q., Effect of ultrasonic treatment on the graft reaction between soy protein isolate and gum acacia and on the physicochemical properties of conjugates. J. Agric. Food Chem. 2010, 58 (7), 4494-9. (11) Martins, S. I. F. S.; Jongen, W. M. F.; Boekel, M. A. J. S. V., A review of Maillard reaction in food and implications to kinetic modelling. Trends Food Sci. Technol. 2000, 11 (9–10), 364-373. (12) Sebeková, K.; Somoza, V., Dietary advanced glycation endproducts (AGEs) and their health effects - PRO. Mol. Nutr. Food Res. 2007, 51 (9), 1079-1084. (13) Poulsen, M. W.; Hedegaard, R. V.; Andersen, J. M.; Courten, B. D.; Bügel, S.; Nielsen, J.; Skibsted, L. H.; Dragsted, L. O., Advanced glycation endproducts in food and their effects on health. Food Chem. Toxicol. 2013, 60 (10), 10. (14) Shi, W.-H.; Sun, W.-W.; Yu, S.-J.; Zhao, M.-M., Study on the characteristic of bovine serum albumin-glucose model system, treated by ultrasonic. Food Res. Int. 2010, 43 (8), 2115-2118. (15) Zhao, C. B.; Zhou, L. Y.; Liu, J. Y.; Zhang, Y.; Chen, Y.; Wu, F., Effect of ultrasonic pretreatment on physicochemical characteristics and rheological properties of soy protein/sugar Maillard reaction products. J. Food Sci. Technol. 2016, 53 (5), 2342. (16) Stanic-Vucinic, D.; Prodic, I.; Apostolovic, D.; Nikolic, M.; Velickovic, T. C., Structure and antioxidant activity of beta-lactoglobulin-glycoconjugates obtained by high-intensity-ultrasound-induced Maillard reaction in aqueous model systems under neutral conditions. Food Chem. 2013, 138 (1), 590-9. (17) Corzo-Martinez, M.; Montilla, A.; Megias-Perez, R.; Olano, A.; Moreno, F. J.; Villamiel, M., Impact of high-intensity ultrasound on the formation of lactulose and Maillard reaction glycoconjugates. Food Chem. 2014, 157, 186-92. (18) Zhang, Q. T.; Tu, Z. C.; Wang, H.; Huang, X. Q.; Fan, L. L.; Bao, Z. Y.; Xiao, H., Functional properties and structure changes of soybean protein isolate after subcritical 23



463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504

water treatment. J. Food Sci. Technol. 2015, 52 (6), 3412-21. (19) Zhang, Q.-T.; Tu, Z.-C.; Xiao, H.; Wang, H.; Huang, X.-Q.; Liu, G.-X.; Liu, C.-M.; Shi, Y.; Fan, L.-L.; Lin, D.-R., Influence of ultrasonic treatment on the structure and emulsifying properties of peanut protein isolate. Food Bioprod. Process. 2014, 92 (1), 30-37. (20) Tu, Z.-c.; Zhong, B.-z.; Wang, H., Identification of glycated sites in ovalbumin under freeze-drying processing by liquid chromatography high-resolution mass spectrometry. Food Chem. 2017, 226, 1-7. (21) Zhang, Q.; Tu, Z.; Wang, H.; Huang, X.; Shi, Y.; Sha, X.; Xiao, H., Improved glycation after ultrasonic pretreatment revealed by high-performance liquid chromatography-linear ion trap/Orbitrap high-resolution mass spectrometry. J. Agric. Food Chem. 2014, 62 (12), 2522-30. (22) Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227 (5259), 680-685. (23) Huang, X.; Tu, Z.; Xiao, H.; Wang, H.; Zhang, L.; Hu, Y.; Zhang, Q.; Niu, P., Characteristics and antioxidant activities of ovalbumin glycated with different saccharides under heat moisture treatment. Food Res. Int. 2012, 48 (2), 866-872. (24) Davies, M. J.; Truscott, R. J., Photo-oxidation of proteins and its role in cataractogenesis. J. Photochem. Photobiol. B: Biol. 2001, 63 (1), 114-125. (25) Seckler, R.; Jaenicke, R., Protein folding and protein refolding. FASEB J. 1992, 6 (8), 2545-52. (26) Pallarès, I.; Vendrell, J.; Avilés, F. X.; Ventura, S., Amyloid fibril formation by a partially structured intermediate state of α-chymotrypsin. J. Mol. Biol. 2004, 342 (1), 321-331. (27) Altoe', P.; Bernardi, F.; Garavelli, M.; Orlandi, G.; Negri, F., Solvent effects on the vibrational activity and photodynamics of the green fluorescent protein chromophore: A quantum-chemical study. J. Am. Chem. Soc. 2005, 127 (11), 3952-3963. (28) Wang, H.; Tu, Z. C.; Liu, G. X.; Zhang, L.; Chen, Y., Identification and quantification of the phosphorylated ovalbumin by high resolution mass spectrometry under dry-heating treatment. Food Chem. 2016, 210, 141. (29) Huang, X.; Tu, Z.; Wang, H.; Zhang, Q.; Shi, Y.; Xiao, H., Increase of Ovalbumin Glycation by the Maillard Reaction after Disruption of the Disulfide Bridge Evaluated by Liquid Chromatography and High Resolution Mass Spectrometry. J. Agric. Food Chem. 2013, 61 (9), 2253. (30) Liu, J.; Tu, Z. C.; Shao, Y. H.; Wang, H.; Liu, G. X.; Sha, X. M.; Zhang, L.; Yang, P., Improved antioxidant activity and glycation of α-lactalbumin after ultrasonic pretreatment revealed by high-resolution mass spectrometry. J. Agric. Food Chem. 2017. (31) Stanic-Vucinic, D.; Stojadinovic, M.; Atanaskovic-Markovic, M.; Ognjenovic, J.; Gronlund, H.; van Hage, M.; Lantto, R.; Sancho, A. I.; Velickovic, T. C., Structural changes and allergenic properties of beta-lactoglobulin upon exposure to high-intensity ultrasound. Mol. Nutr. Food Res. 2012, 56 (12), 1894-905. 24


Page 24 of 34

Page 25 of 34


505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520

(32) Dugaiczyk, A.; Law, S. W.; Dennison, O. E., Nucleotide sequence and the encoded amino acids of human serum albumin mRNA. Proc. Natl. Acad. Sci. USA 1982, 79 (1), 71. (33) Carter, D. C.; Ho, J. X., Structure of serum albumin. Adv. Protein Chem. 1994, 45 (6), 153-176. (34) Mennella, C.; Visciano, M.; Napolitano, A.; Del Castillo, M. D.; Fogliano, V., Glycation of lysine‐containing dipeptides. J. Pept. Sci. 2006, 12 (4), 291-296. (35) Militello, V.; Vetri, V.; Leone, M., Conformational changes involved in thermal aggregation processes of bovine serum albumin. Biophys. Chem. 2003, 105 (1), 133-141. (36) Huang, X.; Tu, Z.; Wang, H.; Zhang, Q.; Hu, Y.; Zhang, L.; Niu, P.; Shi, Y.; Xiao, H., Glycation promoted by dynamic high pressure microfluidisation pretreatment revealed by high resolution mass spectrometry. Food Chem. 2013, 141 (3), 3250-9. (37) Togashi, D. M.; Ryder, A. G.; O'Shaughnessy, D., Monitoring local unfolding of bovine serum albumin during denaturation using steady-state and time-resolved fluorescence spectroscopy. J. Fluoresc. 2010, 20 (2), 441-452.

521

25



Figure Captions:

Figure 1. SDS-PAGE profiles (A) and the bottom image of solution (B) of BSA with ribose under ultrasonic treatment for different times (M: molecular weight marker; N, 5, 10, 15, 20, 25, 30, and 35: NU-BSAR, U-BSAR-5, U-BSAR-10, U-BSAR-15, U-BSAR-20, U-BSAR-25, U-BSAR-30, and U-BSAR-35).

Figure 2. UV absorption spectra (A) and intrinsic fluorescence emission spectra (B) of BSA with ribose under ultrasonic treatment for different times.

Figure 3. The MS spectra of the representative glycated peptides of U-BSAR-35. (A) peptide 179-188 at m/z 619.79622+, (B) peptide 355-364 at m/z 467.21813+, (C) peptide 419-427 at m/z 535.76152+, and (D) peptide 570-580 at m/z 433.55173+. The identified peptides are labelled using residue numbers. Glycation is indicated by double headed arrows. The m/z differences between the glycated and unglycated peptides are indicated above the arrows.

Figure 4. The MS spectra (A) and HCD MS/MS spectrum with peak at m/z 467.28432+ and 533.78182+ (B, C) of glycated peptide (546-552) of U-BSAR-35. The identified


Page 26 of 34

Page 27 of 34


peptides are labelled using residue numbers. Glycation is indicated by double headed arrows. The m/z differences of between the glycated and unglycated peptides are indicated above the arrows. The sequence of each peptide is shown at the top of the mass spectra. b and y ions are shown in the mass spectra, and two glycation sites are remarked by different colors.

Figure 5. Ribbon diagram (A) of the glycated BSA (PDB 4F5S) and partial amino acid residues (B). The lysine residues are colour coded as follows: grey = framework of BSA; red = high content of glycated amino acid residues of BSA under ultrasonication with ribose; green = low content of glycated amino acid residues of BSA under ultrasonication with ribose. In the partial ribbon diagram, the colour code is as follows: yellow = lysine residues; red = hydrophilic amino acid residues; blue = hydrophobic amino acid residues; white = neutral amino acid residues.



Page 28 of 34

Table:

Table 1. Glycated peptides of U-BSAR-35 peptide location

m/z

∆ppm

sequencea

glycated peptide m/z

DSP

glycated site

58-65

487.7248 2+

2.36

(Q)C(CAM)PFDEHVK(L)

553.2448 2+

2.08

K65

127-136

563.7724 2+

0.0265

(F)LSHKDDSPDL(P)

629.7944 2+

0.98

K130

178-188

676.3385

2+

742.3591

2+

4.12

K183

179-188

619.7962

2+

0.867

(L)YYANKYNGVF(Q)

685.8169

2+

6.57

K183

180-188

538.2642 2+

0.374

(Y)YANKYNGVF(Q)

604.2857 2+

4.44

K183

243-251

541.2882 2+

1.34

(L)SQKFPKAEF(V)

607.3092 2+

4.87

K245/K248

316-331

861.4533 2+

2.16

(E)VEKDAIPENLPPLTAD(F)

927.4737 2+

2.99

K318

355-364

467.2181

3+

32.61

R359/R360

370-380

2.22

K374

3.54

K420

27.70

K547, K548

1.52

K580

15.87

K587

1.19

(L)LYYANKYNGVF(Q)

3+

0.577

(L)YEYSRRHPEY(A)

511.2647

653.8727 2+

1.22

(L)LRLAKEYEATL(E)

719.8940 2+

419-427

535.7615 2+

0.394

(F)EKLGEYGFQ(N)

601.7823 2+

546-552

401.2629

2+

570-580

433.5517

3+

587-598

646.3049 2+ a

-0.925

(Q)IKKQTAL(V)

2+

467.2843 /533.7818 3+

-3.38

(T)VMENFVAFVDK(C)

477.5657

2.35

(D)KEAC(CAM)FAVEGPKL(V)

712.3259 2+

CAM refers to the cystine residue alkylated by carbamidomethyl.


2+

Page 29 of 34


Figure Graphics:

Figure 1. SDS-PAGE profiles (A) and the bottom image of solution (B) of BSA with ribose under ultrasonic treatment for different times (M: molecular weight marker; N, 5, 10, 15, 20, 25, 30, and 35: NU-BSAR, U-BSAR-5, U-BSAR-10, U-BSAR-15, U-BSAR-20, U-BSAR-25, U-BSAR-30, and U-BSAR-35).



Abs

0.75

Page 30 of 34

A U-BSAR-35 U-BSAR-15 U-BSAR-5 U-BSAR-25 U-BSAR-20 U-BSAR-10 NU-BSAR U-BSAR-30

0.50

0.25 260

270 280 Wavelength (nm)

290

2800

Fluorescence intensity (a.u.)

B 2400 U-BSAR-5 U-BSAR-10 U-BSAR-15 U-BSAR-20 NU-BSAR U-BSAR-25 U-BSAR-30 U-BSAR-35

2000

1600

1200 320

330

340 350 Wavelength (nm)

360

370

Figure 2. UV absorption spectra (A) and intrinsic fluorescence emission spectra (B) of BSA with ribose under ultrasonic treatment for different times.


Page 31 of 34


Figure 3. The MS spectra of the representative glycated peptides of U-BSAR-35. (A) peptide 179-188 at m/z 619.79622+, (B) peptide 355-364 at m/z 467.21813+, (C) peptide 419-427 at m/z 535.76152+, and (D) peptide 570-580 at m/z 433.55173+. The identified peptides are labelled using residue numbers. Glycation is indicated by double headed arrows. The m/z differences between the glycated and unglycated peptides are indicated above the arrows.



Figure 4. The MS spectra (A) and HCD MS/MS spectrum with peak at m/z 467.28432+ and 533.78182+ (B, C) of glycated peptide (546-552) of U-BSAR-35. The identified peptides are labelled using residue numbers. Glycation is indicated by double headed arrows. The m/z differences of between the glycated and unglycated peptides are indicated above the arrows. The sequence of each peptide is shown at the top of the mass spectra. b and y ions are shown in the mass spectra, and two glycation sites are remarked by different colors.


Page 32 of 34

Page 33 of 34


Figure 5. Ribbon diagram (A) of the glycated BSA (PDB 4F5S) and partial amino acid residues (B). The lysine residues are colour coded as follows: grey = framework of BSA; red = high content of glycated amino acid residues of BSA under ultrasonication with ribose; green = low content of glycated amino acid residues of BSA under ultrasonication with ribose. In the partial ribbon diagram, the colour code is as follows: yellow = lysine residues; red = hydrophilic amino acid residues; blue = hydrophobic amino acid residues; white = neutral amino acid residues.



Table of Contents Graphic:


Page 34 of 34

Liquid Chromatography High-Resolution Mass Spectrometry Identifies

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