Article pubs.acs.org/JAFC
Improved Glycation after Ultrasonic Pretreatment Revealed by High-Performance Liquid Chromatography−Linear Ion Trap/Orbitrap High-Resolution Mass Spectrometry Qiuting Zhang,† Zongcai Tu,*,†,§ Hui Wang,†,# Xiaoqin Huang,§ Yan Shi,† Xiaomei Sha,† and Hui Xiao*,⊥ †
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, China College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi 330022, China # Engineering Research Center for Biomass Conversion, Ministry of Education, Nanchang University, Nanchang, Jiangxi 330047, China ⊥ Department of Pathology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461, United States §
ABSTRACT: The glycation extent of bovine serum albumin (BSA) before and after ultrasonication was evaluated by MALDITOF and Orbitrap mass spectrometry. Ultrasonic pretreatment significantly improved the incorporation of galactose to BSA. Prior to ultrasonic pretreatment, only 12 sites (11 lysines and 1 arginine) were glycated, whereas the number of glycation sites was increased to 42, including 39 lysines and 3 arginines, after treatment. Average degree of substitution per peptide molecule of BSA (DSP) was used to evaluate the glycation level for each glycation site. The ultrasonic pretreatment significantly improved the DSP value of all glycation sites. The prevalently promoted glycation by ultrasonic pretreatment suggests that ultrasonication improves glycation through altering the structure of BSA throughout all three domains. An ultrahigh-resolution linear ion trap Orbitrap mass spectrometer facilitates unambiguous localization of glycation sites, allowing an in-depth analysis of the nature and extent of protein glycation at the molecular level. High-intensity ultrasonication can greatly improve protein glycation and, therefore, opens new routes to modify the functionality of proteins in a positive way. KEYWORDS: BSA, ultrasound, galactose glycation, DSP, mass spectrometry
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treatment.13 Most of the experiments were focused on improving the glycation degree by employing ultrasonication onto the mixture of the protein and sugars. The cavitation effect induced by ultrasonication can generate transient high temperature and pressure, thereby increasing the glycation reaction rate.14 Under the ultrasonication treatment, proteins may undergo conformational changes as demonstrated by Gulseren et al.15 It is not clear whether the ultrasonication accelerated the Maillard reaction directly or through altering the protein conformation. We hypothesize that proteins undergo conformational changes after being treated by ultrasonication, and subsequently these conformational changes favor their glycation via Maillard reaction. To prove this hypothesis, it is necessary to perform the experiment in a stepwise fashion: first, employ ultrasonication on the sugar-free protein (pretreatment) and then add the sugar-pretreated protein to initiate the Maillard reaction. By comparing the glycation extent with and without the ultrasonic pretreatment, we can determine the role of protein conformational change in the glycation reaction. The glycation extent was evaluated mostly using traditional methods, such as browning intensity, free amino acid content, and fluorescence intensity, and by sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS-PAGE).11,12,16,17 These methods can provide only the changes in the protein
INTRODUCTION Glycation is the first step of Maillard reaction occurring between amino and carbonyl compounds, especially reducing sugars. This nonenzymatic glycation plays an important role in industrial food processing, prolonged storage, or domestic cooking. It contributes greatly in food flavor formation, antioxidative effects, and improvement of protein functional properties.1−5 A well-controlled Maillard reaction ensures the quality of food. The major factors that influence the reaction rate and extent of protein glycation are temperature, water activity, and the nature and amount of reducing sugar.4,6 The reactivity of the reducing sugars was reported in the order aldopentose > aldohexoses > aldoketoses > disaccharides.4,6,7 The orientation of the hydroxyl group can also affect the reactivity; for example, D-galactose was found more reactive than its epimer, D-glucose, when glycating BSA by dry-state heating at 60 °C for 30−120 min.8 Many approaches have been applied to improve the Maillard reaction, including heat, microwave, high pressure, irradiation, pulsed electric field, and dynamic high-pressure microfluidization (DHPM).6,9,10 Ultrasonication is also used to promote the glycation reaction. Stanic-Vucinic et al.11 employed ultrasound to accelerate the Maillard reaction of β-lactoglobulin in aqueous model systems under neutral conditions. The ultrasonic treatment was found to accelerate the BSA−glucose glycation and improve the antioxidant properties.12 The ultrasound treatment could significantly speed the glycation process and obtain a protein−polysaccharide conjugate with superior functionality compared to the one under wet-heating-only © 2014 American Chemical Society
Received: Revised: Accepted: Published: 2522
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Each sample was spotted on at least three individual target positions, and 80 individual spectra of each spot were averaged to produce a mass spectrum. The incorporation ratio (IR), that is, the average number of galactose molecules attached to each albumin molecule, was used to evaluate the degree of protein glycation.25 On the basis of the molecular weight (MW) shift from native BSA to glycated BSA, the IR of galactose to BSA could be deduced as
level, without knowing the glycation site(s) and glycation extent per site in the protein. Mass spectrometry, both electrospray ionization mass spectrometry (ESI-MS) and matrixassisted laser desorption ionization−mass spectrometry (MALDI-MS), allow a detailed analysis of the nature and extent of protein modification at the molecular level.18−20 The recent technical innovations in mass spectrometry, particularly with the advent of high-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD), have enabled unambiguous characterization of peptides with various modifications.21,22 HCD allows the detection of the fragments with low mass, which is often not detectable due to the low mass cutoff in ion trap collision-induced dissociation (CID). ETD produces sufficient c and z ions, particularly for those peptides with relatively high mass carrying multiple charges. In addition, ETD can preserve the modification on the amino acid residue, therefore enabling accurate site localization.23,24 The purpose of this study was to evaluate the ultrasonic pretreatment effect on the galactose glycation of BSA. We compared the galactose glycation progress of BSA with and without ultrasonic pretreatment in the dry state. The glycation sites of the native BSA and ultrasound-BSA were characterized by the combined HCD and ETD fragmentation approach. The ultrasonic pretreatment on BSA resulted in many more glycation sites and a stronger glycation degree. Our investigation clearly revealed the critical role of ultrasonication in improving the extent of protein glycation.
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IR = (MWGA − MWNGA)/162.0528
(1)
where 162.0528 is the MW of galactose attached to albumin, MWGA is the molecular weight of glycated BSA, and MWNGA is the molecular weight of nonglycated BSA. Sample Digestion. Four microliters of the protein and complex solution was added to a 500 μL centrifuge tube containing 90 μL of 50 mM ammonium bicarbonate solution and 9 μL of 100 mM DTT. The sample was incubated at 95 °C for 5 min and then cooled in the ice bath. Eighteen microliters of the alkylation buffer (100 mM iodoacetamide) was added to the tube and incubated in the dark at room temperature for 20 min. Six microliters of 0.1 μg/μL trypsin was added to hydrolyze the protein samples at 37 °C for 4 h. The reaction was then quenched by adding 2 μL of 5% trifluoroacetic acid. Analysis by HPLC-HCD/ETD-MS/MS. A Shimadzu HPLC (Shimadzu, Kyoto Japan), with two LC-10AD pumps, was used to generate a gradient with a 100 μL/min flow rate. Solvent A was 5% acetonitrile in H2O, 0.1% formic acid (FA), whereas solvent B consisted of 95% acetonitrile in H2O, 0.1% FA. For analysis of proteolytic peptides, 40 μL of digested sample was injected onto a 2.0 mm i.d. × 100 mm C18 column (Phenomenex Inc., Torrance, CA, USA). After desalting for 5 min with 5% B, the peptides were eluted at 100 μL/min with a 5−10% gradient for 1 min, 10−40% for 20 min, and 40−95% for 2 min. The effluent was infused into an LTQQrbitrap Velos mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA) for MS/MS analysis to identify protein glycation. The decision tree methods were enabled for data acquisition. The settings for the decision tree were as follows: ETD was performed instead of HCD if the charge state was 3 and m/z was 7 times. In addition, compared to the native BSA−galactose, the peak width of ultrasonicated BSA was much broader due to the greater incorporation of galactose. With the changes of molecular weight and peak width taken together, MALDI-TOF mass spectra clearly demonstrated that ultrasound pretreatment significantly improved the glycation
Figure 1. MALDI-TOF-MS analysis of ultrasonicated BSA (A); BSAgalactose before ultrasound (B) and after ultrasound pretreatment (C).
Figure 2. ETD MS/MS spectra of the glycated peptide 52−76 with m/z of 606.07235+ (A) and the glycated peptide 225−239 at m/z of 619.00593+ (B). The sequence of each peptide is shown on top of the peptide. The determined glycation sites are indicated by a line with galactose. The c and z ions are indicated by the numbers and lines. 2524
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Figure 3. Mass spectra of the peptides with multiple glycation sites: (A) peptide 262−285; (B) peptide 348−377. The determined glycation sites are indicated by residue numbers. The mass differences are indicated with numbers and arrows.
glycation, three additional ions were found at m/z 624.10985+, 657.10985+, and 689.52055+, representing one, two, and three glycated forms of this peptide, respectively. Similarly, all three potential glycation sites in the peptide 348−377 were also found to be glycated at their respective sites 350, 362, and 375 (Figure 3B). It is possible for a certain peptide, although exhibiting the mass shift corresponding to monoglycation, to actually contain two (or more) glycated sites. The same modification occurring at different sites generated a mixture of peptides with the same molecular weight. Figure 4 shows one such example. The ion peak with m/z 638.34833+ was found to elute between 4.5 and 5.4 min. The mass of this peptide is calculated as 1912.0201, which is very close to that of the singly glycated peptide 218 LSQKFPKAEFVEVTK232 (Δm = 0.93 ppm). This peptide contains two possible glycation sites, Lys221 and Lys224. The ETD fragmentation of the ion peak at an elution time of 4.92 min suggests that the glycation occurred on Lys221 with the Mascot score of 55 (Figure 4A). At a little earlier elution time of 4.87 min, an ion peak with m/z 479.01224+ appeared and was selected for ETD fragmentation. The mass of the peptide is 1912.0157, also matching the peptide 218−232 with monoglycation (Δm = −1.34 ppm). The tandem mass spectrum of this ion peak indicates that the glycation mostly occurred on Lys224, instead of Lys221, with the Mascot score of 58 (Figure 4B). The results highlight the importance of the efficient ETD fragmentation on the determination of the modification sites. Due to the high similarity between these two peptides, the two mass spectra are almost indistinguishable except for a few fragment ions. The key fragment ions to distinguish the Lys221 modified peptide from the Lys224 modified peptide are c4 (m/z 636.3538) and z9 (m/ z1197.6163). The appearance of a c4 ion in the former peptide
extent of BSA. To fully understand the effect of ultrasound effect on the glycation extent of BSA, it is essential to obtain a detailed map of the glycation sites of BSA with and without ultrasound pretreatment. Such detailed changes in glycation could not be revealed by MALDI-TOF measurement of the intact molecular weight. To determine glycation sites and glycation extent per peptide, we performed tandem mass spectrometry including HCD and ETD after trypsin digestion. Glycation Site Determination. Figure 2 shows the determination of glycation sites Lys64 and Lys232 by MS/ MS. Both sites were found to be glycated in the native and ultrasonicated BSA. Lys64 is identified in peptide 52TCVADESHAGCEKSLHTLFGDELCK76. The mass of the peptide (3025.3256) matched very well with that of the glycated form (2863.2575 + 162.0528 = 3025.3103), with only a 5 ppm difference. The ETD MS/MS of the peptide generated a series of c and z ions (c1−c13 and z2−z14), unambiguously confirming the sequence of the peptide (Figure 2A). The location of the glycation (Lys64) was obtained by the mass difference between c12 and c13 ions, which is the combined mass of lysine and galactose. It is also confirmed by the mass difference between z12 and z13. Similarly, Lys232 was identified as the glycation site from the ETD fragments of the glycated peptide with m/z of 619.00593+ (Figure 2B). The consecutive c and z ions ensure the positive identification of the peptide sequence and glycation site. It should be noted that when an arginine or lysine is modified by glycation, trypsin will not be able to cleave at this particular residue, generating a peptide with a miss-cleavage. Therefore, it is common to observe dually and triply glycated peptides, especially when the glycation degree is high. Figure 3A shows the peptide 262−285, which contains three possible glycation sites at lysine 273, 275, and 280, respectively. The m/z before glycation at charge state 5 was calculated as 592.2848; after 2525
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Figure 4. ETD MS/MS spectra of the singly glycated peptide 218−232 at different elution times and charge states: (A) RT = 4.92 min, charge state of 3 with m/z 638.34833+; (B) RT = 4.87 min, charge state of 4 with m/z 479.01224+. The MS/MS results indicated these two peptides are not identical, although they showed the same mass. The former is glycated at the position of 221 and the latter at the position of 224 as indicated by the colored lysine residues. The c and z ions are indicated by numbers and lines.
freedom of the amine group by steric effects from neighboring atoms, it is not surprising to see the much reduced glycation on arginine relative to lysine. Our glycation results are consistent with previous studies in which the glycation predominantly occurred on lysine but not on arginine.7,8,29 It is worth emphasizing that arginine glycation has not been observed either in our previous study or in the other group’s dry-heating of BSA.8,29 The high-resolution mass accuracy and the versatile fragmentation methods (HCD + ETD) offered by LTQOrbitrap are the major contributions for finding the glycation on arginines because it enables unambiguous identification of the glycation sites, especially for those peptides containing multiple glycation sites. Ultrasonication on the Glycation Extent of BSA. The glycation extent of a protein can be evaluated by (1) the number of the sites that are glycated and (2) the glycation extent per glycation site. When the number of glycation sites is not adequate to assess the glycation extent of the protein, the glycation extent described by DSP can be applied for additional information. In our case, we aim to compare the difference in glycation before and after ultrasonic pretreatment. The effect of this treatment has been clearly demonstrated by the drastically
and a z9 ion in the latter provides unambiguous identification of the location of the glycation. Table 1 shows the sequence and glycation sites of all glycated peptides under both conditions, determined by combined accurate mass and tandem mass spectrometry. BSA contains a total of 82 potential glycation sites including 59 lysines and 23 arginines. Before ultrasonic pretreatment, BSA was found to be glycated at only 12 sites (11K + R), including Lys64, Lys127, K173, Lys204, Lys232, Arg256, Lys280, Lys316, Lys396, Lys439, Lys524, and Lys544. After ultrasonic pretreatment, 42 sites including 39 lysines and 3 arginines were glycated. In addition to the 12 sites in untreated BSA, Lys20, Lys51, Lys76, Lys93, Lys131, Lys136, Lys211, Lys221, Lys224, Lys239, Lys261, Lys273, Lys275, Lys312, Lys322, Lys350, Lys362, Lys375, Lys388, Lys413, Lys465, Lys471, Lys474, Lys499, Lys504, Lys520, Lys523, Lys563, Arg81, and Arg444 were found to be glycated after the ultrasonic pretreatment. The majority of the glycated sites occurred on lysines, as represented by 11 of 59 (19%) and 39 of 59 (66%) lysines in untreated and treated BSA, respectively. In contrast, only 1 of 23 arginines was glycated in native BSA, and only 2 more were found to be glycated after ultrasonication. Given the reduced 2526
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Table 1. Summary of Amino Acid Residues Modified by Galactose in both Native and Ultrasonicated BSA [M + H]+
a
peptide location
m/z (glycated)
obsd
theor
Δ
sequencea
glycated siteb
11−41 42−64 52−76 65−81 77−144 115−136 132−143 144−180 199−208 209−217 218−232 225−239 233−261 240−273 262−285 274−285 286−322 317−335 348−377
777.79505+ 693.32214+ 606.07235+ 527.77404+ 782.34886+ 707.85464+ 579.30933+ 780.03716+ 453.21923+ 388.55263+ 479.01384+ 619.00593+ 591.61886+ 840.57265+ 689.52115+ 565.61673+ 882.22245+ 821.72003+ 816.36955+
3883.9388 2769.2595 3025.3253 2107.0671 4688.0493 2827.3891 1734.9062 4674.1786 1356.6357 1162.6360 1912.0263 1853.9959 3543.6694 4197.8265 3442.5692 1693.8283 4406.0757 2462.1381 4076.8109
3883.9131 2769.2473 3025.3103 2107.0620 4688.0411 2827.3738 1734.8981 4674.1657 1356.6344 1162.6346 1912.0193 1853.9874 3543.6644 4197.8347 3442.5565 1693.8266 4406.0570 2462.1277 4076.7952
0.0257 0.0121 0.0149 0.0051 0.0082 0.0153 0.0027 0.0129 0.0014 0.0014 0.0070 0.0086 0.0050 −0.0083 0.0037 0.0016 0.0187 0.0103 0.0157
(R)FKDLGEEHFKGLVLIAFSQYLQQC*PFDEHVK(L) (K)LVNELTEFAKTC*VADESHAGC*EK(S) (K)TC*VADESHAGC*EKSLHTLFGDELC*K(V) (K)SLHTLFGDELC*KVASLR(E) (K) VASLRETYGDMADC*C*EKQEPERNEC*FLSHKDDSPDLPK(L) (K)LKPDPNTLC*DEFKADEKKFWGK(Y) (K)KFWGKYLYEIAR(R) (R)RHPYFYAPELLYYANKYNGVFQEC*C*QAEDKGAC*LLPK(I) (R)C*ASIQKFGER(A) (R)ALKAWSVAR(L) (R)LSQKFPKAEFVEVTK(L) (K)AEFVEVTKLVTDLTK(V) (K)LVTDLTKVHKEC*C*HGDLLEC*ADDRADLAK(Y) (K)VHKEC*C*HGDLLEC*ADDRADLAKYIC*DNQDTISSK(L) (K)YIC*DNQDTISSKLKEC*C*DKPLLEK(S) (K)LKEC*C*DKPLLEK(S) (K)SHC*IAEVEKDAIPENLPPLTADFAEDKDVC*KNYQEAK(D) (K)NYQEAKDAFLGSFLYEYSR(R) (R)LAKEYEATLEEC*C*AKDDPHAC*YSTVFDKLK(H)
378−409 389−409 413−427 436−458 459−474 472−499 484−504 500−523 505−523 524−533 538−556 545−573
995.25094+ 897.76403+ 601.33403+ 759.34704+ 508.27684+ 873.42524+ 659.06924+ 606.70285+ 610.56134+ 435.59503+ 787.72763+ 868.39184+
3976.9746 2690.2701 1800.9801 3033.3591 2029.0782 3489.6718 2632.2475 3028.4774 2438.2160 1303.7632 2360.1610 3469.5383
3976.9629 2690.2646 1800.9833 3033.3551 2029.0765 3489.6578 2632.2367 3028.4740 2438.2039 1303.7599 2360.1457 3469.5186
0.0029 0.0055 −0.0032 0.0039 0.0016 0.0140 0.0108 0.0035 0.0121 0.0033 0.0152 0.0197
(K)HLVDEPQNLIKQNC*DQFEK(L) (K)QNC*DQFEKLGEYGFQNALIVR(Y) (R)KVPQVSTPTLVEVSR(S) (R)C*C*TKPESERMPC*TEDYLSLILNR(L) (R)LC*VLHEKTPVSEKVTK(C) (K)VTKC*C*TESLVNRRPC*FSALTPDETYVPK(A) (R)RPC*FSALTPDETYVPKAFDEK(L) (K)AFDEKLFTFHADIC*TLPDTEKQIK(K) (K)LFTFHADIC*TLPDTEKQIK(K) (K)KQTALVELLK(H) (K)ATEEQLKTVMENFVAFVDK(C) (K)TVMENFVAFVDKC*C*AADDKEAC*FAVEGPK(L)
K20 K51 K64 K76 K93/R98 K127/K131 K136 K173 K204 K211 K221/K224 K232 K239 R256/K261 K273, K280 K275/K280 K312/K316 K322 K350 + K362 + K375 K388 K396 K413 K439/ R444 K465/K471 K474 K499 K504 K520/K523 K524 K544 K563
C* means the cystine residue alkylated by carbamidomethyl. bThe additional glycation sites after ultrasonic pretreatment are indicated in bold.
The accurate mass and MS/MS determined the ion peak with m/z 564.98793+ is the peptide 225−239. An ion peak appeared at the same elution time with the mass addition of 162.0528 Da, indicating one galactose molecule was added to the peptide. The relative abundance of the glycated form of this peptide increased from 25.57 to 100% after ultrasonication, whereas the relative abundance of the unglycated form of this peptide decreased from 100 to 45.99% after ultrasonication. This indicates that the ultrasonication increased the glycation degree of this peptide. Some other peptides containing glycation site(s) without ultrasonic pretreatment are also shown Figure 5, including 115−136 and 538−556 (Figure 5A,D). The mass spectra of these peptides after ultrasonication are shown underneath (purple trace). Comparison of each mass spectral pair depicted the changes of the degree of glycation of each peptide, with all of the peptides showing improved glycation after ultrasonication. Figure 6 shows the DSP for all of the glycated peptides with and without ultrasonic pretreatment. On the basis of the DSP value, K64 and K204 are the most reactive galactose glycation sites in native BSA with DSP close to 0.8. The ultrasonic pretreatment further increased their DSP to almost full extent at 1.0. For other peptides, most of the DSP values are significantly improved by the ultrasonic pretreatment. It should be
increased glycation sites. However, for those sites modified under both conditions, it is necessary to calculate DSP to evaluate the degree of the glycation. Figure 5 shows mass spectra of the peptides and their corresponding glycated forms before and after ultrasonic pretreatments. The glycated forms of the peptides can be easily determined from the mass difference induced by glycation. Theoretically, if a peptide was monoglycated by galactose, the corresponding m/z peaks with one, two, three, four, or five charges will display a mass increase of 162.0528 Da, with m/z changes of 162.0528, 81.0264, 54.0176, 40.5132, and 32.4106, respectively. For example, the mass spectra of a peptide with a mass of 1356.6357 (m/z 399.2015+3) shown in Figure 5B, the accurate mass and MS/MS determined that it was peptide 199C(carbamidomethyl)ASIQKFGER208. The peak with m/z shift of 54.0178 (m/z 453.2193+3) indicated that one galactose molecule was added to the peptide 199−208, which was again confirmed by MS/MS. The relative abundance of the unglycated form of this peptide was 33.03% before ultrasonication after normalization against the glycated peptide peak. After ultrasonication, the relative abundance decreased to 4.87%, suggesting that the ultrasonication increased the degree of glycation of this peptide. Figure 5C displays another example of this type of ultrasonication effect on the glycation. 2527
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Figure 5. Mass spectra of presentative glycated peptides of BSA after trypsin digestion in both the native and ultrasonicated BSA: peptides 115−136 (A), 199−208 (B), 225−239 (C), and 538−556 (D). Black spectra are the native BSA, and purple spectra are the ultrasonicated BSA.
noted that some peptides, K136 and K474, reached the full glycation with DSP value of 1.0 even though they were not glycated in the native form. This suggests that the protein underwent a structural change under the influence of ultrasonication, exposing some regions of the protein to the solvent and, therefore, gaining more accessibility to the glycation. Because the increased DSP values are distributed all over the protein, this structural perturbation occurred throughout the protein (Figure 6). BSA can be divided into three domains, including domain I comprising residues 1−184, domain II comprising residues 185−377, and domain III comprising residues 378−583.2 The additional glycation sites induced by ultrasonication occurred in all three domains, including 7 sites in domain I (K20, K51, K76, R81, K93, K131, K136), 12 glycation sites in domain II (K211, K221, K224, K239, K261, K273, K275, K312, K322, K350, K362, K375), and 11 sites in
domain III (K388, K413, K465, K471, K474, K499, K504, K520, K523, K563, R444). Taken together, both the increased DSP values and increased glycation sites throughout all three domains suggest that the protein underwent a prevalent structural change in response to ultrasonication. This has been confirmed in a separate conformational study by hydrogen/deuterium exchange and mass spectrometry (data not shown). We have previously performed a similar study on BSA glycation under the influence of DHPM.29 Under both of these two pretreatments, the glycation sites and glycation extent per glycation site are all significantly improved. Despite the similarity, the conformational changes in response to ultrasonication are actually different from those occurring under the influence of DHPM. The conformational changes induced by DHPM are mainly in domains II and III, but not domain I. In contrast, the ultrasonication-triggered conformational changes are distributed throughout all three 2528
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Figure 6. DSP values of glycated sites of native BSA (A) and BSA pretreated by ultrasound (B).
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domains. The applications of these two pretreatments are quite similar; both are used in industry to make emulsions and to produce homogenized liquid.30 The principle of DHPM resides in dividing a pressure stream into two parts, passing each part through a fine orifice, and directing the flows at each other in the microfluidizer interaction chamber. DHPM uses high pressure to guide the flow stream through microchannels in which it generates powerful shear, turbulence, impaction, and cavitation forces that reduce emulsion droplet size.31,32 The major force responsible for ultrasonically induced effects is cavitation, which is the formation and collapse of vapor cavities in a flowing liquid. It is the collapse of these cavities that causes powerful shock waves to radiate throughout the solution, thereby breaking the dispersed liquid. The major difference between these two methods is that ultrasonication is a high-energy and long process, whereas DHPM is a high-energy but short process. In this work, we applied ultrasound with intensity as high as 150 W/cm2 on the BSA for 10 min. The calculated energy density (Ev, J/m3) was 1033 MJ/m3, much higher than the one applied by DHPM at 100 MPa (∼100 MJ/m3).26 It is likely that under the higher energy density of ultrasonication, the protein conformation was perturbed to a much higher extent, causing a structural distortion throughout the whole molecule. Consistent with our results, Guzey et al.33 reported that ultrasound can change the tertiary structure of BSA solution. Ultrasonic pretreatment with temperature control has also been shown to modify the structure of proteins, leading to an increase in surface hydrophobicity.15,34 It should be noted that in this work, the protein was pretreated with ultrasonication in the absence of sugars. The cavitation effect including the intense heat and pressure generated from ultrasonication will be only applied on the protein, but not directly on the glycation process. The improved glycation after ultrasonic pretreatment was therefore solely contributed from the conformational changes of the protein. In conclusion, we employed high-resolution Orbitrap mass spectrometry to investigate the ultrasonication effect on the protein glycation. The results demonstrated that ultrasonication altered the protein structure, favoring glycation, indicating ultrasonication is a promising technology for improving protein functionality in the food industry. This approach opens new routes to modify the functionality of proteins in a positive way, providing a way for a better understanding of the structure− function relationship of the glycated proteins.
AUTHOR INFORMATION
Corresponding Authors
*(H.X.) Phone:1 (718) 430-3469. Fax: 1 (718) 430-8939. E-mail:
[email protected]. *(Z.T.) Phone: +86791 88305938. Fax: +86791 88305938. E-mail:
[email protected]. Funding
This study was supported by the National High Technology Research and Development Program of China (863 Program, No. 2013AA102205), the freedom explore Program of State Key Laboratory of Food Science and Technology of Nanchang University (No. SKLF-ZZB-201310), and the Key Project for Science and Technology Innovation of Jiangxi Province (20124ACB00600). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED BSA, bovine serum albumin; HCD, high-energy C-trap dissociation; ETD, electron transfer dissociation; DHPM, dynamic high-pressure microfluidization; MALDI, matrixassisted laser desorption ionization; ESI, electrospray ionization; DSP, average degree of substitution per peptide molecule; IR, incorporation ratio
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REFERENCES
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Journal of Agricultural and Food Chemistry
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