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Boronic acid modified polyoxometalate-alginate hybrid for the isolation of glycoproteins at neutral environment
Journal Pre-proof Boronic acid modified polyoxometalate-alginate hybrid for the isolation of glycoproteins at neutral environment Wang Xu, Jian-Fang Cao, Yao-Yao Zhang, Yang Shu, Jian-Hua Wang PII:
S0039-9140(19)31253-6
DOI:
https://doi.org/10.1016/j.talanta.2019.120620
Reference:
TAL 120620
To appear in:
Talanta
Received Date: 26 October 2019 Revised Date:
1 December 2019
Accepted Date: 6 December 2019
Please cite this article as: W. Xu, J.-F. Cao, Y.-Y. Zhang, Y. Shu, J.-H. Wang, Boronic acid modified polyoxometalate-alginate hybrid for the isolation of glycoproteins at neutral environment, Talanta (2020), doi: https://doi.org/10.1016/j.talanta.2019.120620. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical abstract
Boronic acid modified polyoxometalate-alginate hybrid for the isolation of glycoproteins at neutral environment
Wang Xu, Jian-Fang Cao, Yao-Yao Zhang, Yang Shu*, Jian-Hua Wang*
Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Shenyang 110819, China
*Corresponding Authors E-mail address: [email protected] (Y. Shu); [email protected](J.H. Wang). Tel: +86 24 83688944; Fax: +86 24 83687659
Abstract Boronate affinity is widely used for the isolation of glycoproteins at alkaline conditions. For further proteomic studies, however, it is of highly importance to perform protein adsorption in neutral medium. For this purpose, we report a novel composite material, i.e., 4-carboxyphenylboronic acid (CPBA) functionalized nickel-substituted polyoxometalate [Ni6(en)3(Tris)(H2O)2(PW9O34)]•nH2O (Ni6PW9)-sodium alginate (SA) hybrid. The abundant oxygen atoms in the hybrid (shortly termed as CPBA-Ni6PW9/SA) significantly reduce the pKa value of CPBA moiety, which well facilitates the selective adsorption of glycoproteins at neutral environment (at pH 7.0). The moiety of sodium alginate (SA) in the hybrid further improves the isolation/enrichment capacity for glycoproteins through hydrophilic interaction. The adsorption efficiency of Immunoglobulin G (IgG, 1.0 mL, 100 µg mL-1) by 1.0 mg CPBA-Ni6PW9/SA hybrid reaches up to 91%, and 1.0 mL of Tris-HCl buffer (100 mmol L-1) provides an elution efficiency of 82%. The adsorption behavior of IgG fits Langmuir adsorption model, offering a maximum adsorption capacity of 495 mg g-1. The CPBA-Ni6PW9/SA hybrid is practically applied for the enrichment of glycoproteins from human serum. SDS-PAGE assay result indicates that approximately 92% serum albumin is eliminated and high-purity IgG is obtained. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis clearly demonstrated that after enriching with CPBA-Ni6PW9/SA, 81 glycoproteins are identified and 79.4% recognition selectivity is achieved.
Keywords: polyoxometalate, sodium alginate, boric acid, glycoprotein, pKa
1. Introduction Glycoprotein is one of the most important post–translational modification proteins which plays crucial role in the fields of signal transduction [1, 2], cell adhesion [3], and endocytosis [4]. The previous studies have demonstrated that glycoprotein has been an essential biomarker for the diagnosis or treatment of diseases [1, 5, 6]. Mass spectrometry has been demonstrated to be the most powerful technique for obtaining comprehensive information of proteomics [7], while tremendous progress should be accomplished to identify the inherent low abundance glycoproteins due to the interference of high abundance non-glycoproteins in complex biological samples [8]. Therefore, the development of efficient approaches for the enrichment of glycoproteins is of great importance. A number of common strategies based various materials, e.g., lectin-based affinity materials [9], hydrazide chemistry materials [10] and antibody-based materials [11], are employed to isolate glycoproteins from biological samples. While their applications are still limited due to the facts that lectins generally show restrictive selectivity to glycoproteins, antibodies exhibit poor stability and their preparations are usually difficult. The reaction of hydrazide chemistry is time-consuming which increases the risk of degeneration of the target object. Recently, boronic acid-based materials and hydrophilic interaction-based materials have attracted extensive attentions for the selective enrichment of glycoproteins/glycopeptides. A number of prominent advantages are identified for boronate affinity materials, e.g., pH-dependent reversible capture/release, outstanding adsorption/enrichment efficiency, and sensitive recognition capabilities toward cis-diol containing compounds [12, 13]. Usually, boronic acid ligands undergo conversion from the trigonal form (pHpKa) by increasing the pH value of the reaction medium. The tetrahedral form of boronic acid ligand can bind to cis-diol compounds, which is more stable than the trigonal form [14]. Boronic acid ligands generally exhibit pKa value of >8.0, and therefore the adsorption/enrichment of glycoproteins is usually conducted under alkaline conditions
[15-17]. Alkaline environments may confuse the interactions between biomolecules, e.g., proteins and enzymes, which affects the accuracy of protein extraction and increases extraction complexity. So far, there are four main approaches for the reduction of pKa values of boronic acid ligands, i.e., introducing an electron-withdrawing group into the phenyl ring [18], forming an intramolecular B-N coordination by incorporating an amino group adjacent to the boron atom (Wulff-type boronic acids) [19], replacing intramolecular B-N with B-O coordination (improved Wulff-type boronic acids) [20], and building “teamed boronate-affinity” [21]. However, these strategies always involve toxic organic reagents or complicated procedures. Hence, novel protocols for the reduction of pKa value of boronic acid ligand with green reagents via simple procedure are highly desired for the selective isolation of glycoproteins at mild conditions. Transition metal-substituted lacunary polyoxometalates (TMSPs) are one of the most fascinating branches in polyoxometalates (POMs) chemistry. They have attracted significant attentions in the fields of catalysis [22], gas sensors [23] and materials science [24] relying on their fascinating properties of easy modification, excellent structural diversity and abundant oxygen atoms. Recently, TMSPs and TMSP-derivatives become a hot topic in living systems. Ni-functionalized Wells– Dawson POMs (Ni-W-POM) exhibit better effects for depressing the amyloid β aggregation and showing peroxidase-like activity inhibition effects, due to the fact that the defined histidine-chelated binding sites of Ni-W-POM could specifically target the HHQK cluster of amyloid β [25]. A platinum (IV)-substituted POM (PtIV-PW11) is encapsulated with 1,2-distearoy-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), which has been used as a prodrug for human colorectal cancer. The mechanism includes the reduction of PtIV to PtII, and then DNA binds to the PtII-substituted POM with subsequent apoptosis [26]. Besides, TMSPs have showed the potential applications in the enrichment of proteins or peptides. In recent study, [{a-PW11O39Zr(µ-OH)(H2O)}2]8- coated magnetic nanospheres show a high adsorption selectivity toward IgG based on the hydrogen-bonding and electrostatic interactions [27]. CeIV-substituted
polyoxometalate functionalized graphene oxide composite shows an excellent selectivity to phosphoproteins based on metal-affinity interaction (phosphate groups and CeIV) and π-stacking interaction (d-p π bonds of POM and phosphate groups) [28]. It is noted that the up to date literatures are short of applications of POMs-containing metarials in the isolation of glycoproteins. In this work, 4-carboxyphenylboronic acid (CPBA) is grafted on NiII-substituted tri-lacunary Keggin type polyoxometalate (Ni6PW9) with abundant oxygen atoms. Ni6PW9 decreases the pKa value of boronic acid ligands through B–O coordination (improved Wulff-type boronic acid). Sodium alginate (SA) with abundant hydroxyl groups and carboxyl groups is introduced into the Ni6PW9 obtaining the final product named as CPBA-Ni6PW9/SA. The developed material CPBA-Ni6PW9/SA exhibits a favorable adsorption behavior towards glycoproteins under neutral environment. The practical application of CPBA-Ni6PW9/SA is well demonstrated by the enrichment of glycoproteins from human serum samples.
2. Experimental section 2.1 Materials and Chemicals Sodium tungstate dihydrate (Na2WO4·2H2O, >99.5%), sodium alginate (SA), sodium bisulfite (NaHSO3) and 4-carboxyphenylboronic acid (CPBA, >98%) are purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Nickel (II) chloride hexahydrate (NiCl2·6H2O), sodium nitrite (NaNO2), 2-morpholinoethanesulfonic acid (MES), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) are achieved from Aladdin (Shanghai, China). Immunoglobulin G from human serum (IgG, >95%, Mr 150.0 kDa, pI 6.95), transferrin (Trf, >98%, Mr 78 kDa, pI 5.2-5.9), human serum albumin (HSA, Mr 67 kDa, pI 4.7-4.9), ovalbumin (OVA, Mr 44.3 kDa, pI 4.7), bovine serum albumin (BSA, Mr 66.4 kDa, pI 4.9), conalbumin (ConA, 98%, Mr 140 kDa pI 6.8), α-lactalbumin from bovine milk (α-La, >85%, pI 4.2-4.5) and γ-globulin from bovine serum (γ-Glo, Mr 50 kDa, pI 6.3-7.3) are obtained from Sigma-Aldrich (St Louis, USA). Tris(hydroxymethyl)aminomethane (Tris, >99.5%) is the product from Dalian
Meilun Biotechnology Co. Ltd. (Dalian, China). All the reagents are at least of analytical-reagent grade and used without further purification. Deionized water of 18 MΩ cm is used throughout the experiments. Human whole blood is provided by healthy volunteers from the Hospital of Northeastern University. 2.2 Characterizations UV-vis absorption spectra are recorded on a U-3900 UV-vis spectrophotometer (Hitachi High Technologies, Japan). Fourier transform infrared spectra (FT-IR) are recorded on a VERTEX 70 FT-IR spectrophotometer (Brooker, Germany) from 4000 to 400 cm-1. Thermogravimetric analysis is performed on a TGA/DSC3+ analyzer (TGA, METTLER TOLEDO, Switzerland). X-ray diffraction (XRD) patterns are obtained on an Empyrean (BRUKER, Germany) with Cu Kα radiation at λ 1.54 Å. Scanning electron microscopy (SEM) images are recorded on a SU8010 scanning electron microscope (Hitachi High Technologies, Japan). Transmission electron microscopy (TEM) images are achieved by using a Tecnai G220 (FEI, Netherlands). Nitrogen adsorption/desorption experiments are performed on a Autosorb-IQ-MP-C analyzer (Quantachrome, USA). X-ray photoelectron spectroscopy (XPS) measurements are performed on a Thermo Scientific ESCALAB 250Xi electron spectrometer (Thermo Electron, America). Zeta potential measurements for surface charge analysis are performed on a Zetasizer Nano ZS90 (Malvern, UK). 2.3 Preparation of Ni6PW9/SA The PW9 is prepared according to a previous reference [29]. Shortly, 0.30 g PW9, 0.40 g Tris and 0.80 g NiCl2·6H2O are added to 10 mL sodium acetate buffer (0.5 mol L-1, pH 4.8,). The mixture is stirred for 5 min at room temperature, and a green clear solution is formed. Then, 0.30 mL ethylenediamine is added drop-wisely under stirring, and the solution changes into a blue suspension. After that, 0.20 g SA is added and continuously stirred for 1 h. The resulting mixture is sealed into a 50 mL stainless steel reactor with a Teflon liner reacted at 160 °C for 25 h, and then cooling down to room temperature. Subsequently, the resulting product is collected by suction filtration and washed with deionized water. The final brown Ni6PW9/SA powder is obtained after freeze-drying overnight. The Ni6PW9 is prepared as control. The
procedure is similar to that of Ni6PW9/SA except that none SA is added. 2.4 Preparation of CPBA-Ni6PW9/SA hybrid 1.50 g CPBA is dissolved into 30mL of MES buffer (0.1 mol L-1, pH 5.6). 2.97 g EDC and 2.97 g NHS are then added to activate the carboxyl group of CPBA at 40 °C for 45 min, followed by cooling down to room temperature. Subsequently, the above reaction mixture is adjusted to pH 7.0 with 0.1 mol L-1 Na2HPO4 solution. 0.225 g Ni6PW9/SA is added into the mixture following ultrasonic treatment for 5 min and stirring at room temperature for 5 h. The obtained CPBA-Ni6PW9/SA is collected by suction filtration and washed with deionized water for three times. Finally, the CPBA-Ni6PW9/SA powder is dried under freeze-drying overnight. 2.5 Protein adsorption and desorption 1.0 mg of CPBA-Ni6PW9/SA is mixed with 1.0 mL of protein solution at 100 µg mL-1 (in 40 mmol L-1 Britton−Robinson (BR) buffer). The adsorption of protein is then conducted by oscillating for 30 min at room temperature. The CPBA-Ni6PW9/SA with adsorbed proteins is collected by centrifugation at 8000 rpm for 10 min. 1.0 mL of Tris-HCl buffer (100 mmol L-1) is used as the elution reagent to recover the retained proteins with shaking for 30 min, followed by separation of the CPBA-Ni6PW9/SA hybrid by centrifugation at 8000 rpm for 10 min. The protein quantification of supernatant is performed by spectrophotometry using a U-3900 UV-vis spectrophotometer by recording the characteristic absorption at 595 nm after staining with Coomassie brilliant blue (IgG, γ-Glo, OVA, ConA, Trf, HSA, α-La). 2.6 The adsorption isotherms of glycoproteins 0.20 mg of CPBA-Ni6PW9/SA hybrid is used for the adsorption of IgG of various concentrations (within a range of 0.05-0.50 mg mL-1, in 40 mmol L-1 BR buffer, pH 7.0). The adsorption capacity Qe (mg g-1) is calculated according to the equation in the following, where C0 (mg mL-1) is the initial concentration of IgG, Ce (mg mL-1) is the equilibrium concentration of IgG after extraction, V (mL) is the volume of protein solution and m (mg) is the mass of CPBA-Ni6PW9/SA. Two universal adsorption models, i.e., Langmuir model (2) and Freundlich model (3) [30], are adopted to analyze the experimental data as expressed in the following equation,
in which Ce (mg L-1) is the protein equilibrium concentration, Qe (mg g-1) is the amount of adsorbed protein at equilibrium, Qmax (mg g-1) is the adsorption capacity, KL and KF are the adsorption equilibrium constants, and n is the adsorption intensity. =
− 1 10
1 1 1 1 = ∙ + 2 1 log = log + log 3 2.7 Glycoproteins enrichment from human serum 10 µL of human serum is diluted with 990 µL BR buffer (40 mmol L-1, pH 7.0) and incubated with 1.0 mg CPBA-Ni6PW9/SA hybrid for 30 min. After centrifugation at 8000 rpm for 10 min, the supernatant is removed and then the loosely retained portions on the surface of CPBA-Ni6PW9/SA is eliminated by washing for 3 times with BR buffer (40 mmol L-1, pH 7.0). Subsequently, 1.0 mL of Tris-HCl buffer (100 mmol L-1, pH 10.0) is added to release the glycoproteins from CPBA-Ni6PW9/SA for 30 min. After centrifugation at 8000 rpm for 10 min, the stripped proteins are obtained.
3. Results and discussion 3.1 Characterizations of the CPBA-Ni6PW9/SA hybrid The preparation procedures of CPBA-Ni6PW9/SA hybrid are illustrated in Scheme 1. PW9 is modified with Ni2+, SA and Tris in one-pot hydrothermal reaction. The lacunary positions of PW9 are occupied by Ni2+ forming Ni6PW9. The six terminal water ligands on the Ni6PW9 offer the possibility to be replaced by the carboxyl functional groups of SA [31]. Meanwhile, the three hydroxyl groups located on the axial positions of the Ni6 cluster are substituted by the tripodal alcohol ligands of Tris [32]. The obtained Ni6PW9/SA is further functionalized with CPBA through amide reaction between the –NH2 group of Tris and the –COOH group of CPBA. The
final product is denoted as CPBA-Ni6PW9/SA.
Scheme 1. pH dependence of reversible conformational transition of boronic acid group (A) and the schematic illustration for the preparation of the CPBA-Ni6PW9/SA hybrid (B). The morphologies of PW9 and CPBA-Ni6PW9/SA are characterized by SEM and TEM images. As shown in Figure 1A, the size of PW9 uint is ca. 2-8nm in diameter. Figure 1B indicates the linkage of CPBA-Ni6PW9 by SA to form the CPBA-Ni6PW9/SA hybrid. The SEM image of CPBA-Ni6PW9/SA displays a block stacking morphology in Figure 1C.
Figure 1. TEM images of PW9 (A) and CPBA-Ni6PW9/SA (B), SEM image of CPBA-Ni6PW9/SA (C). The inset shows the size-distribution histogram. FT-IR spectra of SA, PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA are shown in Figure 2A. For SA, the bands of O=C‒O asymmetric stretching vibration at 1612 cm-1 and C‒OH deformation vibration at 1417 cm-1 are assigned to the carboxylate groups. Those at 941 and 896 cm-1 are assigned to the C–O stretching vibrations of uronic
acid and α-l-gulopyranuronic asymmetric ring vibration [33]. For PW9, the broad band at 3445 cm-1 is attributed to the O‒H stretching vibration of crystal water, and the asymmetric stretching vibrations of P‒O at 1056 and 1014 cm-1, W=O at 931 cm-1 and W‒O‒W at 885 and 821 cm-1 are recognized [34]. After modification of SA onto the Ni6PW9, the bands of Ni6PW9/SA attributing to the asymmetric stretching vibrations of P‒O, W=O, M‒O‒M (M=W or Ni) red shifted to 1041 and 943 cm-1, 840 cm-1, 796 and 711 cm-1, respectively. In the FT-IR spectrum of CPBA-Ni6PW9/SA, the bands at 1371, 1207 and 1074 cm-1 are ascribed to the stretching vibration of B‒O, the bending vibration of B‒O‒H and the stretching mode of C‒B, respectively. The bands at 1650-1400 cm-1 are the characteristic absorptions of benzene ring of CPBA. That at 1336 cm-1 is the stretching vibration of C‒N of amide group which is formed through amidation interaction between –COOH of CPBA and –NH2 of Ni6PW9/SA [35]. The above results clearly indicated the successful preparation of the CPBA-Ni6PW9/SA hybrid. XRD peaks of CPBA-Ni6PW9/SA have a significant change compared with those of PW9 as shown in Figure 2B. The characteristic peaks of B-α-PW9 at 8.6°, 11.4°, 17.1° disappeared and the intense 2θcharacteristic peaks of A-α-PW9 at 22.3°, 30.7°, 32.1°, 32.9° are retained, which indicates that the B-α-PW9 transforms to A-α-PW9 after hydro-thermal treatment (A and B indicate the types of isomer of α-PW9) [36]. TGA analysis results for PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA hybrid are shown in Figure 2C. The weight loss stage of PW9 is observed with losing about 8.7% within a temperature range of 30-180 °C. This is attributed to the evaporation of lattice water. Ni6PW9/SA shows two continuous stages of weight loss. The first weight loss is ca.10.1% within 30-310 °C, involving the loss of lattice water and coordinated water molecules with Ni2+. The second weight loss stage is due to the thermo-decompositions of SA and Tris at a temperature higher than 310 °C. Three obvious weight loss stages for CPBA-Ni6PW9/SA are observed. The lattice water and coordinated water molecules evaporate at 30-180 °C. Then CPBA decomposes with 40% of weight loss. The last stage of weight loss is the thermo-decompositions of SA and Tris (>310 °C) which is similar to the TGA curve of Ni6PW9/SA. These results
indicate that the CPBA-Ni6PW9/SA hybrid possesses favorable thermo-stability and is suitable for enrichment of glycoproteins. From the N2 adsorption–desorption isotherms, the calculated surface areas of Ni6PW9/SA and CPBA-Ni6PW9/SA are 59.7 and 15.9 m2 g−1 respectively by the Brunauer–Emmett–Teller (BET) method (Figure 2D). The surface area of CPBA-Ni6PW9/SA is decreased after functionalization with CPBA, which is attributed to that CPBA modification occurs on the outer surface of Ni6PW9/SA. The insert shows the BJH pore-size distribution curves of Ni6PW9/SA and CPBA-Ni6PW9/SA, which displays wide range distribution. This is indicates that the block aggregating provides inhomogeneous sizes of slit-shaped pores [37].
Figure 2. (A) FT-IR spectra of SA, PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA. (B) XRD patterns of PW9 and CPBA-Ni6PW9/SA. (C) TGA curves of PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA. (D) Nitrogen adsorption−desorption isotherms and pore size distribution curves (inset) of Ni6PW9/SA (a) and CPBA-Ni6PW9/SA (b). The full range XPS spectrum of CPBA-Ni6PW9/SA is showed in Figure 3A. The six peaks of Ni 2p (857.1 eV), O 1s (533.1 eV), N 1s (402.1 eV), C 1s (285.1 eV), B
1s (192.1 eV) and W 4f (37.1 eV) are clearly observed. High-resolution XPS spectra of W 4f and Ni 2p are shown in Figure S1A and Figure S1B. The narrow range characteristic peaks of W 4f at 35.9 eV and 38.0 eV correspond to W 4f5/2 and W 4f7/2, respectively [38]. The characteristic peaks at 861.9 eV and 880.1 eV of Ni 2p are deconvoluted into one spin-orbit coupling and two shakeup satellites. The doublet peaks at 856.2 eV (Ni 2p3/2) and 873.9 eV (Ni 2p1/2) showing energy difference gap of 17.7 eV are assigned to Ni2+ phase [39]. High-resolution XPS spectrum of B 1s is shown in Figure 3B, which is deconvoluted into two peaks at 191.2 and 192.1 eV, corresponding to B–C and B–O bond, respectively [35]. The five peaks of C 1s at 284.0, 285.4, 284.7, 286.1 and 288.7 eV are attributed to the C–B bond of CPBA, C– N bond of amide group, C=C/C–C, C–O and C=O bonds of CPBA and SA, respectively (Figure S1C) [35, 40, 41]. These results suggest that the CPBA-Ni6PW9/SA hybrid is prepared as designed.
Figure 3. The full range XPS spectrum of CPBA-Ni6PW9/SA (A) and the narrow range high-resolution XPS spectrum of B 1s (B). pKa values of CPBA and CPBA-Ni6PW9/SA are evaluated with UV-vis absorption spectra. There is a strong absorption at ca. 285 nm for CPBA solution within a range of pH 2.0-8.0, and the absorption band disappears at pH>8.5 (Figure 4A). Based on this observation, the pKa value of CPBA could be speculated within 8.0 and 8.5, which is well consistent with the theoretical value of pKa 8.0 [42]. After modification with Ni6PW9/SA, the absorption band of CPBA-Ni6PW9/SA at ca. 285 nm is reserved within the range of pH 2.0-6.0, and it vanishes at pH＞6.5 (Figure 4B).
Therefore, the pKa value of CPBA-Ni6PW9/SA is in the range of 6.5-7.0, which is obviously reduced in comparison with that of CPBA. The quantitative terminal oxygen atoms at the surface of Ni6PW9 build the B-O coordination with B atom of CPBA resulted in the improved formation of Wulff-type boronic acids, which reduces the pKa value of CPBA within 7.0. 3.2 Selective adsorption of glycoproteins by the CPBA-Ni6PW9/SA hybrid
Figure 4. UV-vis absorption spectra of CPBA (A) and CPBA-Ni6PW9/SA (B) within a wide range of pH 2.0-11.0. BR buffer: 40 mmol L-1. CPBA concentration: 200 µg mL-1. CPBA-Ni6PW9/SA concentration: 100 µg mL-1. A series of glycoprotein models, i.e., IgG, γ-Glo, OVA, ConA and Trf, with two non-glycoprotein models, i.e., HSA and α-La, are chosen to evaluate the adsorption selectivity of Ni6PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA toward glycoproteins. As shown in Figure 5, the adsorption efficiencies for IgG, γ-Glo, OVA, ConA, Trf, HSA and α-La by Ni6PW9 are 30.4%, 3.6%, 19.8%, 0.2%, 41.8%, 63.2% and 58.2%, respectively. Ni6PW9 shows obvious no specific adsorption of proteins. After encapsulation of SA, the adsorption capacities of Ni6PW9/SA toward IgG, γ-Glo, OVA, ConA and Trf are improved to 49.4%, 64.3%, 49.4%, 74.4% and 42.3%. SA contains high density of hydrophilic groups, e.g., hydroxyl and carboxyl groups, which could form a hydrophilic layer in the surrounding area of SA [43]. Hence, the hydrophilic interaction between SA and the glycans of glycoprotein has a great positive effect on the adsorption capacity of glycoproteins. However, the adsorption efficiency of non-glycoproteins is also improved which indicates that Ni6PW9/SA also exhibits no specific adsorption for proteins. Among the amino acid composition of
HSA, 10.2% is glutamic acid and 9.9% is lysine. As for α-La, 8.5% of amino acid is lysine and 9.2% is aspartic acid. These amino acid residues of non-glycoproteins could build hydrogen bonds with not only –NH2 groups of Tris on the axial positions of the Ni6 cluster, but also –OH groups of SA. Thus, the increase on the adsorption efficiency for the non-glycoproteins is virtually not observed with Ni6PW9/SA as the adsorbent. In comparison with Ni6PW9/SA, the adsorption efficiency of glycoproteins by adopting CPBA-Ni6PW9/SA as the adsorbent is further significantly enhanced, especially for IgG and γ-Glo, with adsorption efficiencies of as high as 91.4% and 94.3%, respectively. At the same experimental conditions, however, the adsorption efficiency of non-glycoproteins is obviously reduced. It is reason that the decreasing of hydrogen bonds after the –NH2 of Tris in Ni6PW9/SA reacted with –COOH of CPBA. Therefore, the SA has a barely affection on the adsorption of non-glycoproteins. HSA, as the most abundant non-glycoprotein, its adsorption efficiency is greatly reduced to as low as 5.6%. These results indicate that CPBA makes a great contribution to the adsorption selectivity toward glycoproteins based on the boronate affinity, and SA plays a significant role on the adsorption capability of glycoproteins relying on its hydrophilicity. The CPBA-Ni6PW9/SA hybrid is thus most suitable for the selective enrichment of glycoproteins.
Figure 5. The adsorption efficiencies of Ni6PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA for glycoproteins and non-glycoproteins. Protein solution: 100 µg mL-1, 1.0 mL. Adsorbent: 1.0 mg. Adsorption time: 30 min. BR buffer: 40 mmol L-1, pH 7.0.
3.3 pH dependent adsorption of proteins by the CPBA-Ni6PW9/SA hybrid Boronic acid moiety usually performs favourable affinity with glycoprotein at pH>pKa. In this study, pKa value of the CPBA moiety on CPBA-Ni6PW9/SA is decreased to 6.5-7.0 by Ni6PW9. To confirm the practicability, a series of glycoprotein models, i.e., IgG, γ-Glo, OVA, ConA and Trf, are used to investigate the adsorption behaviors of CPBA-Ni6PW9/SA at various pHs (pH 6.0, 7.0, 8.0). The results show that maximum adsorption efficiencies are achieved at pH 7.0 for all the glycoproteins (Figure 6). The adsorption efficiencies for IgG, γ-Glo, OVA, ConA, and Trf are 91.4%, 94.3%, 66.0%, 73.3% and 57.3%, respectively. This observation well verifies the feasibility for the selective adsorption of glycoproteins with CPBA-Ni6PW9/SA in a neutral condition. The boronic acid moiety could form more stable boronic ester bond with cis-diol groups of the glycoprotein at pH 7.0 and 8.0, which are higher than the pKa of CPBA-Ni6PW9/SA. However, at pH 8.0, the adsorption efficiencies of IgG, γ-Glo, OVA, ConA and Trf are 67.1%, 51.9%, 49.0% 19.2% and 19.4% respectively, which are obviously lower than those achieved at pH 7.0. At the pH close to the isoelectric point (pI) of a protein, the protein presents a most compact conformation and a high structural stability with a minimal charge. As a result, the electrostatic repulsion between the proteins is the lowest and it is beneficial for the adsorption of proteins [44]. Glycoproteins, i.e., IgG (pI 6.95), γ-Glo (pI 6.3-7.3), OVA (pI 4.7), ConA (pI 6.8) and Trf (pI 5.2-5.9), are all negatively charged at pH 8.0. The CPBA-Ni6PW9/SA hybrid is also negatively charged within the range of pH 4.0-11.0 as illustrated in the zeta potential diagram (Figure S2). Electrostatic repulsion between the protein species, as well as between the adsorbed proteins and the adsorbent inhibits the combination of glycoproteins and CPBA-Ni6PW9/SA hybrid. Therefore, the adsorption capacity of glycoprotein at pH 8.0 is inferior to that at pH 7.0. These results illustrate that the best adsorption performance to glycoprotein is obtained in a neutral buffer.
Figure 6. pH-dependent adsorption behaviors of glycoproteins (IgG, γ-Glo, OVA, ConA, Trf) with CPBA-Ni6PW9/SA as the adsorbent. Protein solution: 100 µg mL-1, 1.0 mL. CPBA-Ni6PW9/SA: 1.0 mg. Adsorption time: 30 min. BR buffer: 40 mmol L-1, pH 7.0. The influence of ionic strength on the adsorption efficiency is further investigated. As showed in Figure S3A, there is no significant change in the adsorption efficiency of IgG onto the CPBA-Ni6PW9/SA hybrid when increasing NaCl concentration within the range of 0-0.5 mol L-1. In addition, the adsorption efficiency reaches saturation rapidly when the adsorption time is more than 30 min (Figure S3B). Considering that a low salt concentration is convenient and favorable for the treatment of real biological samples, 40 mmol L-1 BR buffer of pH 7.0 without NaCl is adopted to prepare the sample solution. An adsorption time of 30 min is conducted in the following experiments. 3.4 The adsorption isotherm of CPBA-Ni6PW9/SA hybrid IgG and OVA are high abundant glycoproteins in human serum and chicken egg white, respectively. IgG and OVA are chose as glycoprotein models to investigate the dynamic adsorption behaviors with wide range concentrations of 0.05-0.50 mg mL-1 in BR buffer solution (pH 7.0) (Figure 7). CPBA-Ni6PW9/SA exhibits a high adsorption capacity of 495.8 and 373.3 mg g-1 for IgG and OVA, respectively. In order to investigate the adsorption mechanism of glycoproteins onto CPBA-Ni6PW9/SA, two universal adsorption models, i.e., Langmuir model (2) and Freundlich model (3)
[30], are adopted to analyze the experimental data. Obviously, the adsorption behaviors of IgG and OVA all fit well with the Langmuir adsorption model. The result indicates that the adsorption behaviors of IgG and OVA onto the CPBA-Ni6PW9/SA hybrid are monolayer adsorption (Langmuir model) instead of multilayer adsorption (Freundlich model).
Figure 7. The adsorption isotherms of IgG and OVA onto the CPBA-Ni6PW9/SA hybrid. Protein solution: 0.05-0.50 mg mL-1, 1.0 mL. CPBA-Ni6PW9/SA: 1.0 mg. Adsorption time: 30 min. BR buffer: 40 mmol L-1, pH 7.0. In order to illustrate the adsorption performance of CPBA-Ni6PW9/SA, Table 1 compares the adsorption equilibrium time, adsorption capacity and optimal adsorption pH value for some boronate affinity materials with IgG or OVA as model protein. On the whole, CPBA-Ni6PW9/SA hybrid not only displays a fast adsorption equilibrium, but also could be employed for the selective isolation of glycoproteins in mild conditions. It is seen that a favorable adsorption capacity is achievedwith the CPBA-Ni6PW9/SA hybrid with respect to other boronic acid functional materials at a neutral pH.
Table 1. The comparison of adsorption performances of various boronate affinity materials on glycoproteins. Adsorbent
Glycoprotein model
Adsorption equilibrium time
Adsorption capacity (mg g-1)
pH
Ref.
Fe3O4@SiO2-BA
OVA
>4 h
<14
8.5
[45]
Fe3O4@pPMA-MPBA
OVA
4h
337.3
9.0
[46]
CA-g-P(AM-co-BA)
OVA
3h
─
9.0/7.4
[47]
SiO2-MIPs
OVA
60 min
243.4
8.5
[48]
D-MIPs
OVA
30 min
138.9
8.5
[49]
PW12@TiO2-Si(Et)Si/Pba
IgG
30 min
348.5
9.0
[50]
Fe3O4@SiO2@UiO-PBA
OVA
30 min
327.3
7.4
[12]
Fe3O4-FPBA(MMIPs)
OVA
25 min
51.2
7.4
[51]
Fe3O4@PGMA-TBA/MIPs
OVA
50 min
190.7
7.4
[52]
CPBA-Ni6PW9/SA
IgG
30 min
495.8
7.0
This
OVA
373.3
work
3.5 Recovery of the adsorbed IgG from CPBA-Ni6PW9/SA hybrid The recovery of the adsorbed IgG from the CPBA-Ni6PW9/SA hybrid is shown in Figure 8 with BR buffer and Tris-HCl buffer as eluents. Although boronate affinity materials could adsorb glycoproteins at alkaline conditions and the retained glycoproteins could be released at acidic conditions, hardly any IgG is recovered from the CPBA-Ni6PW9/SA hybrid even at pH 2.0. Besides, an elution efficiency of 36.8% is achieved at pH 10.0 in this study. IgG (pI 6.95) is positively charged at pH 2.0. Therefore, the electrostatic attraction between protein–surface (CPBA-Ni6PW9/SA) makes the elution of IgG from CPBA-Ni6PW9/SA hybrid is difficult. At pH 10.0, the electrostatic repulsion weakens the adsorption of IgG onto the CPBA-Ni6PW9/SA hybrid. Considering the strong hydrophilic interaction between CPBA-Ni6PW9/SA hybrid and IgG, Tris-HCl buffer is selected as the eluent. Tris contains a tripodal alcohol ligand and exhibits a stronger hydrophilic interaction with CPBA-Ni6PW9/SA
than IgG, which releases competitively the adsorbed IgG from the surface of CPBA-Ni6PW9/SA. At pH 10.0, by increasing the concentration of Tris-HCl buffer from 50 to 100 mmol L-1, the elution efficiency is increased from 71.3% to 82.1%. Therefore, in the future studies, 100 mmol L-1 of Tris-HCl (pH 10.0) is chosen as the eluent for recovering the retained glycoproteins from the CPBA-Ni6PW9/SA hybrid.
Figure 8. The recoveries of the adsorbed IgG from CPBA-Ni6PW9/SA hybrid with various eluents. Protein solution: 100 µg mL-1, 1.0 mL. CPBA-Ni6PW9/SA: 1.0 mg. Adsorption time: 30 min. BR buffer: 40 mmol L-1. Tris-HCl buffer: 50 and 100 mmol L-1. The volume of the stripping reagent: 1.0 mL. Stripping time: 30 min 3.6 Enrichment of glycoproteins from human serum with CPBA-Ni6PW9/SA The practicability of CPBA-Ni6PW9/SA hybrid for the adsorption of glycoproteins is carried out by isolating IgG from human serum. SDS polyacrylamide gel electrophoresis (SDS-PAGE) assay results are shown in Figure 9. Ten protein bands of the diluted human serum sample are identified which are mainly corresponding to HSA (66.4 kDa), IgG heavy chain (50 kDa), and IgG light chain (25 kDa) (lane 2). After recovering the proteins with Tris-HCl buffer solution, only the bands of IgG (lane 4) are observed. It is well consistent with the bands of IgG standard solution (lane 5). Image J software is used to analyze the intensity of the serum albumin at lane 2 and lane 4 [53]. It can be obtained by calculation that approximately 92% of the high abundance non-glycoprotein, i.e., serum albumin, is
removed during the adsorption process, and IgG is selectively separated from the human serum.
Figure 9. SDS-PAGE analysis of human serum before and after treatment with CPBA-Ni6PW9/SA. Lane 1: protein marker (kDa), lane 2: serum sample after 100-fold dilution without pretreatment by CPBA-Ni6PW9/SA, lane 3: 100-fold diluted serum sample after treating with CPBA-Ni6PW9/SA, lane 4: protein recovered from CPBA-Ni6PW9/SA, lane 5: IgG standard solution (150 µg mL-1). Moreover, LC-MS/MS is further adopted to evaluate the practicality of CPBA-Ni6PW9/SA hybrid in the isolation of glycoproteins. CPBA-Ni6PW9/SA hybrid is used to enrich glycoproteins from human serum under neutral environment and the absorbed proteins are recovered as described in the above sections. Through trypsin digestion and PNGase F deglycosylation, the obtained peptides are analyzed by LC-MS/MS. To ensure high confidence identifications, a minimum of two unique peptides per protein is adopted to filter proteins. There are 81 glycoproteins identified after treatment with the CPBA-Ni6PW9/SA hybrid, providing a recognition selectivity of 79.4%. The identified glycoproteins data are recorded in Table S1, suggesting that the CPBA-Ni6PW9/SA hybrid exhibits a favorable affinity to glycoproteins.
4. Conclusions A novel hybrid material for enrichment of glycoproteins is prepared by modifying NiII-substituted lacunary Keggin-type polyoxometalate with sodium
alginate via a one-pot hydrothermal approach, and subsequently functionalized with 4-carboxyphenylboronic acid through amide reaction. The obtained CPBA-Ni6PW9/SA hybrid significantly decreases the pKa value of CPBA to 6.5-7.0, attributing to the B-O coordination of B atom in CPBA and abundant oxygen atoms of Ni6PW9. Thus high adsorption selectivity towards glycoproteins is realized at mild environment of pH 7.0. This adsorption behavior of CPBA-Ni6PW9/SA is not only attributed to the boronate affinity interaction, but also due to the hydrophilic interaction. The present study offers a novel strategy for the reduction of the pKa value of boronic acid moiety, which is able to enrich glycoproteins from complex biological samples under neutral conditions.
5. Acknowledgements This work is financially supported by the Natural Science Foundation of China (21974018, 21727811, 21575020). Fundamental Research Funds for the Central Universities (N170505002, N170504017, N170507001).
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Highlights 1. The pKa value of 4-carboxyphenylboronic acidis successfully decreased via modification with a nickel-substituted polyoxometalate. 2. The well-designed hybrid is suitable for the selective adsorption of glycoproteins in neutral environment (pH 7.0). 3. The novel hybrid possesses excellent adsorption behavior toward glycoproteins based on boronate affinity interaction and hydrophilic interaction. 4. Highly specific enrichment of glycoproteins from human serum is achieved with the CPBA-Ni6PW9/SA hybrid.
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0039-9140(19)31253-6
DOI:
https://doi.org/10.1016/j.talanta.2019.120620
Reference:
TAL 120620
To appear in:
Talanta
Received Date: 26 October 2019 Revised Date:
1 December 2019
Accepted Date: 6 December 2019
Please cite this article as: W. Xu, J.-F. Cao, Y.-Y. Zhang, Y. Shu, J.-H. Wang, Boronic acid modified polyoxometalate-alginate hybrid for the isolation of glycoproteins at neutral environment, Talanta (2020), doi: https://doi.org/10.1016/j.talanta.2019.120620. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical abstract
Boronic acid modified polyoxometalate-alginate hybrid for the isolation of glycoproteins at neutral environment
Wang Xu, Jian-Fang Cao, Yao-Yao Zhang, Yang Shu*, Jian-Hua Wang*
Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Shenyang 110819, China
*Corresponding Authors E-mail address: [email protected] (Y. Shu); [email protected](J.H. Wang). Tel: +86 24 83688944; Fax: +86 24 83687659
Abstract Boronate affinity is widely used for the isolation of glycoproteins at alkaline conditions. For further proteomic studies, however, it is of highly importance to perform protein adsorption in neutral medium. For this purpose, we report a novel composite material, i.e., 4-carboxyphenylboronic acid (CPBA) functionalized nickel-substituted polyoxometalate [Ni6(en)3(Tris)(H2O)2(PW9O34)]•nH2O (Ni6PW9)-sodium alginate (SA) hybrid. The abundant oxygen atoms in the hybrid (shortly termed as CPBA-Ni6PW9/SA) significantly reduce the pKa value of CPBA moiety, which well facilitates the selective adsorption of glycoproteins at neutral environment (at pH 7.0). The moiety of sodium alginate (SA) in the hybrid further improves the isolation/enrichment capacity for glycoproteins through hydrophilic interaction. The adsorption efficiency of Immunoglobulin G (IgG, 1.0 mL, 100 µg mL-1) by 1.0 mg CPBA-Ni6PW9/SA hybrid reaches up to 91%, and 1.0 mL of Tris-HCl buffer (100 mmol L-1) provides an elution efficiency of 82%. The adsorption behavior of IgG fits Langmuir adsorption model, offering a maximum adsorption capacity of 495 mg g-1. The CPBA-Ni6PW9/SA hybrid is practically applied for the enrichment of glycoproteins from human serum. SDS-PAGE assay result indicates that approximately 92% serum albumin is eliminated and high-purity IgG is obtained. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis clearly demonstrated that after enriching with CPBA-Ni6PW9/SA, 81 glycoproteins are identified and 79.4% recognition selectivity is achieved.
Keywords: polyoxometalate, sodium alginate, boric acid, glycoprotein, pKa
1. Introduction Glycoprotein is one of the most important post–translational modification proteins which plays crucial role in the fields of signal transduction [1, 2], cell adhesion [3], and endocytosis [4]. The previous studies have demonstrated that glycoprotein has been an essential biomarker for the diagnosis or treatment of diseases [1, 5, 6]. Mass spectrometry has been demonstrated to be the most powerful technique for obtaining comprehensive information of proteomics [7], while tremendous progress should be accomplished to identify the inherent low abundance glycoproteins due to the interference of high abundance non-glycoproteins in complex biological samples [8]. Therefore, the development of efficient approaches for the enrichment of glycoproteins is of great importance. A number of common strategies based various materials, e.g., lectin-based affinity materials [9], hydrazide chemistry materials [10] and antibody-based materials [11], are employed to isolate glycoproteins from biological samples. While their applications are still limited due to the facts that lectins generally show restrictive selectivity to glycoproteins, antibodies exhibit poor stability and their preparations are usually difficult. The reaction of hydrazide chemistry is time-consuming which increases the risk of degeneration of the target object. Recently, boronic acid-based materials and hydrophilic interaction-based materials have attracted extensive attentions for the selective enrichment of glycoproteins/glycopeptides. A number of prominent advantages are identified for boronate affinity materials, e.g., pH-dependent reversible capture/release, outstanding adsorption/enrichment efficiency, and sensitive recognition capabilities toward cis-diol containing compounds [12, 13]. Usually, boronic acid ligands undergo conversion from the trigonal form (pH
[15-17]. Alkaline environments may confuse the interactions between biomolecules, e.g., proteins and enzymes, which affects the accuracy of protein extraction and increases extraction complexity. So far, there are four main approaches for the reduction of pKa values of boronic acid ligands, i.e., introducing an electron-withdrawing group into the phenyl ring [18], forming an intramolecular B-N coordination by incorporating an amino group adjacent to the boron atom (Wulff-type boronic acids) [19], replacing intramolecular B-N with B-O coordination (improved Wulff-type boronic acids) [20], and building “teamed boronate-affinity” [21]. However, these strategies always involve toxic organic reagents or complicated procedures. Hence, novel protocols for the reduction of pKa value of boronic acid ligand with green reagents via simple procedure are highly desired for the selective isolation of glycoproteins at mild conditions. Transition metal-substituted lacunary polyoxometalates (TMSPs) are one of the most fascinating branches in polyoxometalates (POMs) chemistry. They have attracted significant attentions in the fields of catalysis [22], gas sensors [23] and materials science [24] relying on their fascinating properties of easy modification, excellent structural diversity and abundant oxygen atoms. Recently, TMSPs and TMSP-derivatives become a hot topic in living systems. Ni-functionalized Wells– Dawson POMs (Ni-W-POM) exhibit better effects for depressing the amyloid β aggregation and showing peroxidase-like activity inhibition effects, due to the fact that the defined histidine-chelated binding sites of Ni-W-POM could specifically target the HHQK cluster of amyloid β [25]. A platinum (IV)-substituted POM (PtIV-PW11) is encapsulated with 1,2-distearoy-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), which has been used as a prodrug for human colorectal cancer. The mechanism includes the reduction of PtIV to PtII, and then DNA binds to the PtII-substituted POM with subsequent apoptosis [26]. Besides, TMSPs have showed the potential applications in the enrichment of proteins or peptides. In recent study, [{a-PW11O39Zr(µ-OH)(H2O)}2]8- coated magnetic nanospheres show a high adsorption selectivity toward IgG based on the hydrogen-bonding and electrostatic interactions [27]. CeIV-substituted
polyoxometalate functionalized graphene oxide composite shows an excellent selectivity to phosphoproteins based on metal-affinity interaction (phosphate groups and CeIV) and π-stacking interaction (d-p π bonds of POM and phosphate groups) [28]. It is noted that the up to date literatures are short of applications of POMs-containing metarials in the isolation of glycoproteins. In this work, 4-carboxyphenylboronic acid (CPBA) is grafted on NiII-substituted tri-lacunary Keggin type polyoxometalate (Ni6PW9) with abundant oxygen atoms. Ni6PW9 decreases the pKa value of boronic acid ligands through B–O coordination (improved Wulff-type boronic acid). Sodium alginate (SA) with abundant hydroxyl groups and carboxyl groups is introduced into the Ni6PW9 obtaining the final product named as CPBA-Ni6PW9/SA. The developed material CPBA-Ni6PW9/SA exhibits a favorable adsorption behavior towards glycoproteins under neutral environment. The practical application of CPBA-Ni6PW9/SA is well demonstrated by the enrichment of glycoproteins from human serum samples.
2. Experimental section 2.1 Materials and Chemicals Sodium tungstate dihydrate (Na2WO4·2H2O, >99.5%), sodium alginate (SA), sodium bisulfite (NaHSO3) and 4-carboxyphenylboronic acid (CPBA, >98%) are purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Nickel (II) chloride hexahydrate (NiCl2·6H2O), sodium nitrite (NaNO2), 2-morpholinoethanesulfonic acid (MES), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) are achieved from Aladdin (Shanghai, China). Immunoglobulin G from human serum (IgG, >95%, Mr 150.0 kDa, pI 6.95), transferrin (Trf, >98%, Mr 78 kDa, pI 5.2-5.9), human serum albumin (HSA, Mr 67 kDa, pI 4.7-4.9), ovalbumin (OVA, Mr 44.3 kDa, pI 4.7), bovine serum albumin (BSA, Mr 66.4 kDa, pI 4.9), conalbumin (ConA, 98%, Mr 140 kDa pI 6.8), α-lactalbumin from bovine milk (α-La, >85%, pI 4.2-4.5) and γ-globulin from bovine serum (γ-Glo, Mr 50 kDa, pI 6.3-7.3) are obtained from Sigma-Aldrich (St Louis, USA). Tris(hydroxymethyl)aminomethane (Tris, >99.5%) is the product from Dalian
Meilun Biotechnology Co. Ltd. (Dalian, China). All the reagents are at least of analytical-reagent grade and used without further purification. Deionized water of 18 MΩ cm is used throughout the experiments. Human whole blood is provided by healthy volunteers from the Hospital of Northeastern University. 2.2 Characterizations UV-vis absorption spectra are recorded on a U-3900 UV-vis spectrophotometer (Hitachi High Technologies, Japan). Fourier transform infrared spectra (FT-IR) are recorded on a VERTEX 70 FT-IR spectrophotometer (Brooker, Germany) from 4000 to 400 cm-1. Thermogravimetric analysis is performed on a TGA/DSC3+ analyzer (TGA, METTLER TOLEDO, Switzerland). X-ray diffraction (XRD) patterns are obtained on an Empyrean (BRUKER, Germany) with Cu Kα radiation at λ 1.54 Å. Scanning electron microscopy (SEM) images are recorded on a SU8010 scanning electron microscope (Hitachi High Technologies, Japan). Transmission electron microscopy (TEM) images are achieved by using a Tecnai G220 (FEI, Netherlands). Nitrogen adsorption/desorption experiments are performed on a Autosorb-IQ-MP-C analyzer (Quantachrome, USA). X-ray photoelectron spectroscopy (XPS) measurements are performed on a Thermo Scientific ESCALAB 250Xi electron spectrometer (Thermo Electron, America). Zeta potential measurements for surface charge analysis are performed on a Zetasizer Nano ZS90 (Malvern, UK). 2.3 Preparation of Ni6PW9/SA The PW9 is prepared according to a previous reference [29]. Shortly, 0.30 g PW9, 0.40 g Tris and 0.80 g NiCl2·6H2O are added to 10 mL sodium acetate buffer (0.5 mol L-1, pH 4.8,). The mixture is stirred for 5 min at room temperature, and a green clear solution is formed. Then, 0.30 mL ethylenediamine is added drop-wisely under stirring, and the solution changes into a blue suspension. After that, 0.20 g SA is added and continuously stirred for 1 h. The resulting mixture is sealed into a 50 mL stainless steel reactor with a Teflon liner reacted at 160 °C for 25 h, and then cooling down to room temperature. Subsequently, the resulting product is collected by suction filtration and washed with deionized water. The final brown Ni6PW9/SA powder is obtained after freeze-drying overnight. The Ni6PW9 is prepared as control. The
procedure is similar to that of Ni6PW9/SA except that none SA is added. 2.4 Preparation of CPBA-Ni6PW9/SA hybrid 1.50 g CPBA is dissolved into 30mL of MES buffer (0.1 mol L-1, pH 5.6). 2.97 g EDC and 2.97 g NHS are then added to activate the carboxyl group of CPBA at 40 °C for 45 min, followed by cooling down to room temperature. Subsequently, the above reaction mixture is adjusted to pH 7.0 with 0.1 mol L-1 Na2HPO4 solution. 0.225 g Ni6PW9/SA is added into the mixture following ultrasonic treatment for 5 min and stirring at room temperature for 5 h. The obtained CPBA-Ni6PW9/SA is collected by suction filtration and washed with deionized water for three times. Finally, the CPBA-Ni6PW9/SA powder is dried under freeze-drying overnight. 2.5 Protein adsorption and desorption 1.0 mg of CPBA-Ni6PW9/SA is mixed with 1.0 mL of protein solution at 100 µg mL-1 (in 40 mmol L-1 Britton−Robinson (BR) buffer). The adsorption of protein is then conducted by oscillating for 30 min at room temperature. The CPBA-Ni6PW9/SA with adsorbed proteins is collected by centrifugation at 8000 rpm for 10 min. 1.0 mL of Tris-HCl buffer (100 mmol L-1) is used as the elution reagent to recover the retained proteins with shaking for 30 min, followed by separation of the CPBA-Ni6PW9/SA hybrid by centrifugation at 8000 rpm for 10 min. The protein quantification of supernatant is performed by spectrophotometry using a U-3900 UV-vis spectrophotometer by recording the characteristic absorption at 595 nm after staining with Coomassie brilliant blue (IgG, γ-Glo, OVA, ConA, Trf, HSA, α-La). 2.6 The adsorption isotherms of glycoproteins 0.20 mg of CPBA-Ni6PW9/SA hybrid is used for the adsorption of IgG of various concentrations (within a range of 0.05-0.50 mg mL-1, in 40 mmol L-1 BR buffer, pH 7.0). The adsorption capacity Qe (mg g-1) is calculated according to the equation in the following, where C0 (mg mL-1) is the initial concentration of IgG, Ce (mg mL-1) is the equilibrium concentration of IgG after extraction, V (mL) is the volume of protein solution and m (mg) is the mass of CPBA-Ni6PW9/SA. Two universal adsorption models, i.e., Langmuir model (2) and Freundlich model (3) [30], are adopted to analyze the experimental data as expressed in the following equation,
in which Ce (mg L-1) is the protein equilibrium concentration, Qe (mg g-1) is the amount of adsorbed protein at equilibrium, Qmax (mg g-1) is the adsorption capacity, KL and KF are the adsorption equilibrium constants, and n is the adsorption intensity. =
− 1 10
1 1 1 1 = ∙ + 2 1 log = log + log 3 2.7 Glycoproteins enrichment from human serum 10 µL of human serum is diluted with 990 µL BR buffer (40 mmol L-1, pH 7.0) and incubated with 1.0 mg CPBA-Ni6PW9/SA hybrid for 30 min. After centrifugation at 8000 rpm for 10 min, the supernatant is removed and then the loosely retained portions on the surface of CPBA-Ni6PW9/SA is eliminated by washing for 3 times with BR buffer (40 mmol L-1, pH 7.0). Subsequently, 1.0 mL of Tris-HCl buffer (100 mmol L-1, pH 10.0) is added to release the glycoproteins from CPBA-Ni6PW9/SA for 30 min. After centrifugation at 8000 rpm for 10 min, the stripped proteins are obtained.
3. Results and discussion 3.1 Characterizations of the CPBA-Ni6PW9/SA hybrid The preparation procedures of CPBA-Ni6PW9/SA hybrid are illustrated in Scheme 1. PW9 is modified with Ni2+, SA and Tris in one-pot hydrothermal reaction. The lacunary positions of PW9 are occupied by Ni2+ forming Ni6PW9. The six terminal water ligands on the Ni6PW9 offer the possibility to be replaced by the carboxyl functional groups of SA [31]. Meanwhile, the three hydroxyl groups located on the axial positions of the Ni6 cluster are substituted by the tripodal alcohol ligands of Tris [32]. The obtained Ni6PW9/SA is further functionalized with CPBA through amide reaction between the –NH2 group of Tris and the –COOH group of CPBA. The
final product is denoted as CPBA-Ni6PW9/SA.
Scheme 1. pH dependence of reversible conformational transition of boronic acid group (A) and the schematic illustration for the preparation of the CPBA-Ni6PW9/SA hybrid (B). The morphologies of PW9 and CPBA-Ni6PW9/SA are characterized by SEM and TEM images. As shown in Figure 1A, the size of PW9 uint is ca. 2-8nm in diameter. Figure 1B indicates the linkage of CPBA-Ni6PW9 by SA to form the CPBA-Ni6PW9/SA hybrid. The SEM image of CPBA-Ni6PW9/SA displays a block stacking morphology in Figure 1C.
Figure 1. TEM images of PW9 (A) and CPBA-Ni6PW9/SA (B), SEM image of CPBA-Ni6PW9/SA (C). The inset shows the size-distribution histogram. FT-IR spectra of SA, PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA are shown in Figure 2A. For SA, the bands of O=C‒O asymmetric stretching vibration at 1612 cm-1 and C‒OH deformation vibration at 1417 cm-1 are assigned to the carboxylate groups. Those at 941 and 896 cm-1 are assigned to the C–O stretching vibrations of uronic
acid and α-l-gulopyranuronic asymmetric ring vibration [33]. For PW9, the broad band at 3445 cm-1 is attributed to the O‒H stretching vibration of crystal water, and the asymmetric stretching vibrations of P‒O at 1056 and 1014 cm-1, W=O at 931 cm-1 and W‒O‒W at 885 and 821 cm-1 are recognized [34]. After modification of SA onto the Ni6PW9, the bands of Ni6PW9/SA attributing to the asymmetric stretching vibrations of P‒O, W=O, M‒O‒M (M=W or Ni) red shifted to 1041 and 943 cm-1, 840 cm-1, 796 and 711 cm-1, respectively. In the FT-IR spectrum of CPBA-Ni6PW9/SA, the bands at 1371, 1207 and 1074 cm-1 are ascribed to the stretching vibration of B‒O, the bending vibration of B‒O‒H and the stretching mode of C‒B, respectively. The bands at 1650-1400 cm-1 are the characteristic absorptions of benzene ring of CPBA. That at 1336 cm-1 is the stretching vibration of C‒N of amide group which is formed through amidation interaction between –COOH of CPBA and –NH2 of Ni6PW9/SA [35]. The above results clearly indicated the successful preparation of the CPBA-Ni6PW9/SA hybrid. XRD peaks of CPBA-Ni6PW9/SA have a significant change compared with those of PW9 as shown in Figure 2B. The characteristic peaks of B-α-PW9 at 8.6°, 11.4°, 17.1° disappeared and the intense 2θcharacteristic peaks of A-α-PW9 at 22.3°, 30.7°, 32.1°, 32.9° are retained, which indicates that the B-α-PW9 transforms to A-α-PW9 after hydro-thermal treatment (A and B indicate the types of isomer of α-PW9) [36]. TGA analysis results for PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA hybrid are shown in Figure 2C. The weight loss stage of PW9 is observed with losing about 8.7% within a temperature range of 30-180 °C. This is attributed to the evaporation of lattice water. Ni6PW9/SA shows two continuous stages of weight loss. The first weight loss is ca.10.1% within 30-310 °C, involving the loss of lattice water and coordinated water molecules with Ni2+. The second weight loss stage is due to the thermo-decompositions of SA and Tris at a temperature higher than 310 °C. Three obvious weight loss stages for CPBA-Ni6PW9/SA are observed. The lattice water and coordinated water molecules evaporate at 30-180 °C. Then CPBA decomposes with 40% of weight loss. The last stage of weight loss is the thermo-decompositions of SA and Tris (>310 °C) which is similar to the TGA curve of Ni6PW9/SA. These results
indicate that the CPBA-Ni6PW9/SA hybrid possesses favorable thermo-stability and is suitable for enrichment of glycoproteins. From the N2 adsorption–desorption isotherms, the calculated surface areas of Ni6PW9/SA and CPBA-Ni6PW9/SA are 59.7 and 15.9 m2 g−1 respectively by the Brunauer–Emmett–Teller (BET) method (Figure 2D). The surface area of CPBA-Ni6PW9/SA is decreased after functionalization with CPBA, which is attributed to that CPBA modification occurs on the outer surface of Ni6PW9/SA. The insert shows the BJH pore-size distribution curves of Ni6PW9/SA and CPBA-Ni6PW9/SA, which displays wide range distribution. This is indicates that the block aggregating provides inhomogeneous sizes of slit-shaped pores [37].
Figure 2. (A) FT-IR spectra of SA, PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA. (B) XRD patterns of PW9 and CPBA-Ni6PW9/SA. (C) TGA curves of PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA. (D) Nitrogen adsorption−desorption isotherms and pore size distribution curves (inset) of Ni6PW9/SA (a) and CPBA-Ni6PW9/SA (b). The full range XPS spectrum of CPBA-Ni6PW9/SA is showed in Figure 3A. The six peaks of Ni 2p (857.1 eV), O 1s (533.1 eV), N 1s (402.1 eV), C 1s (285.1 eV), B
1s (192.1 eV) and W 4f (37.1 eV) are clearly observed. High-resolution XPS spectra of W 4f and Ni 2p are shown in Figure S1A and Figure S1B. The narrow range characteristic peaks of W 4f at 35.9 eV and 38.0 eV correspond to W 4f5/2 and W 4f7/2, respectively [38]. The characteristic peaks at 861.9 eV and 880.1 eV of Ni 2p are deconvoluted into one spin-orbit coupling and two shakeup satellites. The doublet peaks at 856.2 eV (Ni 2p3/2) and 873.9 eV (Ni 2p1/2) showing energy difference gap of 17.7 eV are assigned to Ni2+ phase [39]. High-resolution XPS spectrum of B 1s is shown in Figure 3B, which is deconvoluted into two peaks at 191.2 and 192.1 eV, corresponding to B–C and B–O bond, respectively [35]. The five peaks of C 1s at 284.0, 285.4, 284.7, 286.1 and 288.7 eV are attributed to the C–B bond of CPBA, C– N bond of amide group, C=C/C–C, C–O and C=O bonds of CPBA and SA, respectively (Figure S1C) [35, 40, 41]. These results suggest that the CPBA-Ni6PW9/SA hybrid is prepared as designed.
Figure 3. The full range XPS spectrum of CPBA-Ni6PW9/SA (A) and the narrow range high-resolution XPS spectrum of B 1s (B). pKa values of CPBA and CPBA-Ni6PW9/SA are evaluated with UV-vis absorption spectra. There is a strong absorption at ca. 285 nm for CPBA solution within a range of pH 2.0-8.0, and the absorption band disappears at pH>8.5 (Figure 4A). Based on this observation, the pKa value of CPBA could be speculated within 8.0 and 8.5, which is well consistent with the theoretical value of pKa 8.0 [42]. After modification with Ni6PW9/SA, the absorption band of CPBA-Ni6PW9/SA at ca. 285 nm is reserved within the range of pH 2.0-6.0, and it vanishes at pH＞6.5 (Figure 4B).
Therefore, the pKa value of CPBA-Ni6PW9/SA is in the range of 6.5-7.0, which is obviously reduced in comparison with that of CPBA. The quantitative terminal oxygen atoms at the surface of Ni6PW9 build the B-O coordination with B atom of CPBA resulted in the improved formation of Wulff-type boronic acids, which reduces the pKa value of CPBA within 7.0. 3.2 Selective adsorption of glycoproteins by the CPBA-Ni6PW9/SA hybrid
Figure 4. UV-vis absorption spectra of CPBA (A) and CPBA-Ni6PW9/SA (B) within a wide range of pH 2.0-11.0. BR buffer: 40 mmol L-1. CPBA concentration: 200 µg mL-1. CPBA-Ni6PW9/SA concentration: 100 µg mL-1. A series of glycoprotein models, i.e., IgG, γ-Glo, OVA, ConA and Trf, with two non-glycoprotein models, i.e., HSA and α-La, are chosen to evaluate the adsorption selectivity of Ni6PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA toward glycoproteins. As shown in Figure 5, the adsorption efficiencies for IgG, γ-Glo, OVA, ConA, Trf, HSA and α-La by Ni6PW9 are 30.4%, 3.6%, 19.8%, 0.2%, 41.8%, 63.2% and 58.2%, respectively. Ni6PW9 shows obvious no specific adsorption of proteins. After encapsulation of SA, the adsorption capacities of Ni6PW9/SA toward IgG, γ-Glo, OVA, ConA and Trf are improved to 49.4%, 64.3%, 49.4%, 74.4% and 42.3%. SA contains high density of hydrophilic groups, e.g., hydroxyl and carboxyl groups, which could form a hydrophilic layer in the surrounding area of SA [43]. Hence, the hydrophilic interaction between SA and the glycans of glycoprotein has a great positive effect on the adsorption capacity of glycoproteins. However, the adsorption efficiency of non-glycoproteins is also improved which indicates that Ni6PW9/SA also exhibits no specific adsorption for proteins. Among the amino acid composition of
HSA, 10.2% is glutamic acid and 9.9% is lysine. As for α-La, 8.5% of amino acid is lysine and 9.2% is aspartic acid. These amino acid residues of non-glycoproteins could build hydrogen bonds with not only –NH2 groups of Tris on the axial positions of the Ni6 cluster, but also –OH groups of SA. Thus, the increase on the adsorption efficiency for the non-glycoproteins is virtually not observed with Ni6PW9/SA as the adsorbent. In comparison with Ni6PW9/SA, the adsorption efficiency of glycoproteins by adopting CPBA-Ni6PW9/SA as the adsorbent is further significantly enhanced, especially for IgG and γ-Glo, with adsorption efficiencies of as high as 91.4% and 94.3%, respectively. At the same experimental conditions, however, the adsorption efficiency of non-glycoproteins is obviously reduced. It is reason that the decreasing of hydrogen bonds after the –NH2 of Tris in Ni6PW9/SA reacted with –COOH of CPBA. Therefore, the SA has a barely affection on the adsorption of non-glycoproteins. HSA, as the most abundant non-glycoprotein, its adsorption efficiency is greatly reduced to as low as 5.6%. These results indicate that CPBA makes a great contribution to the adsorption selectivity toward glycoproteins based on the boronate affinity, and SA plays a significant role on the adsorption capability of glycoproteins relying on its hydrophilicity. The CPBA-Ni6PW9/SA hybrid is thus most suitable for the selective enrichment of glycoproteins.
Figure 5. The adsorption efficiencies of Ni6PW9, Ni6PW9/SA and CPBA-Ni6PW9/SA for glycoproteins and non-glycoproteins. Protein solution: 100 µg mL-1, 1.0 mL. Adsorbent: 1.0 mg. Adsorption time: 30 min. BR buffer: 40 mmol L-1, pH 7.0.
3.3 pH dependent adsorption of proteins by the CPBA-Ni6PW9/SA hybrid Boronic acid moiety usually performs favourable affinity with glycoprotein at pH>pKa. In this study, pKa value of the CPBA moiety on CPBA-Ni6PW9/SA is decreased to 6.5-7.0 by Ni6PW9. To confirm the practicability, a series of glycoprotein models, i.e., IgG, γ-Glo, OVA, ConA and Trf, are used to investigate the adsorption behaviors of CPBA-Ni6PW9/SA at various pHs (pH 6.0, 7.0, 8.0). The results show that maximum adsorption efficiencies are achieved at pH 7.0 for all the glycoproteins (Figure 6). The adsorption efficiencies for IgG, γ-Glo, OVA, ConA, and Trf are 91.4%, 94.3%, 66.0%, 73.3% and 57.3%, respectively. This observation well verifies the feasibility for the selective adsorption of glycoproteins with CPBA-Ni6PW9/SA in a neutral condition. The boronic acid moiety could form more stable boronic ester bond with cis-diol groups of the glycoprotein at pH 7.0 and 8.0, which are higher than the pKa of CPBA-Ni6PW9/SA. However, at pH 8.0, the adsorption efficiencies of IgG, γ-Glo, OVA, ConA and Trf are 67.1%, 51.9%, 49.0% 19.2% and 19.4% respectively, which are obviously lower than those achieved at pH 7.0. At the pH close to the isoelectric point (pI) of a protein, the protein presents a most compact conformation and a high structural stability with a minimal charge. As a result, the electrostatic repulsion between the proteins is the lowest and it is beneficial for the adsorption of proteins [44]. Glycoproteins, i.e., IgG (pI 6.95), γ-Glo (pI 6.3-7.3), OVA (pI 4.7), ConA (pI 6.8) and Trf (pI 5.2-5.9), are all negatively charged at pH 8.0. The CPBA-Ni6PW9/SA hybrid is also negatively charged within the range of pH 4.0-11.0 as illustrated in the zeta potential diagram (Figure S2). Electrostatic repulsion between the protein species, as well as between the adsorbed proteins and the adsorbent inhibits the combination of glycoproteins and CPBA-Ni6PW9/SA hybrid. Therefore, the adsorption capacity of glycoprotein at pH 8.0 is inferior to that at pH 7.0. These results illustrate that the best adsorption performance to glycoprotein is obtained in a neutral buffer.
Figure 6. pH-dependent adsorption behaviors of glycoproteins (IgG, γ-Glo, OVA, ConA, Trf) with CPBA-Ni6PW9/SA as the adsorbent. Protein solution: 100 µg mL-1, 1.0 mL. CPBA-Ni6PW9/SA: 1.0 mg. Adsorption time: 30 min. BR buffer: 40 mmol L-1, pH 7.0. The influence of ionic strength on the adsorption efficiency is further investigated. As showed in Figure S3A, there is no significant change in the adsorption efficiency of IgG onto the CPBA-Ni6PW9/SA hybrid when increasing NaCl concentration within the range of 0-0.5 mol L-1. In addition, the adsorption efficiency reaches saturation rapidly when the adsorption time is more than 30 min (Figure S3B). Considering that a low salt concentration is convenient and favorable for the treatment of real biological samples, 40 mmol L-1 BR buffer of pH 7.0 without NaCl is adopted to prepare the sample solution. An adsorption time of 30 min is conducted in the following experiments. 3.4 The adsorption isotherm of CPBA-Ni6PW9/SA hybrid IgG and OVA are high abundant glycoproteins in human serum and chicken egg white, respectively. IgG and OVA are chose as glycoprotein models to investigate the dynamic adsorption behaviors with wide range concentrations of 0.05-0.50 mg mL-1 in BR buffer solution (pH 7.0) (Figure 7). CPBA-Ni6PW9/SA exhibits a high adsorption capacity of 495.8 and 373.3 mg g-1 for IgG and OVA, respectively. In order to investigate the adsorption mechanism of glycoproteins onto CPBA-Ni6PW9/SA, two universal adsorption models, i.e., Langmuir model (2) and Freundlich model (3)
[30], are adopted to analyze the experimental data. Obviously, the adsorption behaviors of IgG and OVA all fit well with the Langmuir adsorption model. The result indicates that the adsorption behaviors of IgG and OVA onto the CPBA-Ni6PW9/SA hybrid are monolayer adsorption (Langmuir model) instead of multilayer adsorption (Freundlich model).
Figure 7. The adsorption isotherms of IgG and OVA onto the CPBA-Ni6PW9/SA hybrid. Protein solution: 0.05-0.50 mg mL-1, 1.0 mL. CPBA-Ni6PW9/SA: 1.0 mg. Adsorption time: 30 min. BR buffer: 40 mmol L-1, pH 7.0. In order to illustrate the adsorption performance of CPBA-Ni6PW9/SA, Table 1 compares the adsorption equilibrium time, adsorption capacity and optimal adsorption pH value for some boronate affinity materials with IgG or OVA as model protein. On the whole, CPBA-Ni6PW9/SA hybrid not only displays a fast adsorption equilibrium, but also could be employed for the selective isolation of glycoproteins in mild conditions. It is seen that a favorable adsorption capacity is achievedwith the CPBA-Ni6PW9/SA hybrid with respect to other boronic acid functional materials at a neutral pH.
Table 1. The comparison of adsorption performances of various boronate affinity materials on glycoproteins. Adsorbent
Glycoprotein model
Adsorption equilibrium time
Adsorption capacity (mg g-1)
pH
Ref.
Fe3O4@SiO2-BA
OVA
>4 h
<14
8.5
[45]
Fe3O4@pPMA-MPBA
OVA
4h
337.3
9.0
[46]
CA-g-P(AM-co-BA)
OVA
3h
─
9.0/7.4
[47]
SiO2-MIPs
OVA
60 min
243.4
8.5
[48]
D-MIPs
OVA
30 min
138.9
8.5
[49]
PW12@TiO2-Si(Et)Si/Pba
IgG
30 min
348.5
9.0
[50]
Fe3O4@SiO2@UiO-PBA
OVA
30 min
327.3
7.4
[12]
Fe3O4-FPBA(MMIPs)
OVA
25 min
51.2
7.4
[51]
Fe3O4@PGMA-TBA/MIPs
OVA
50 min
190.7
7.4
[52]
CPBA-Ni6PW9/SA
IgG
30 min
495.8
7.0
This
OVA
373.3
work
3.5 Recovery of the adsorbed IgG from CPBA-Ni6PW9/SA hybrid The recovery of the adsorbed IgG from the CPBA-Ni6PW9/SA hybrid is shown in Figure 8 with BR buffer and Tris-HCl buffer as eluents. Although boronate affinity materials could adsorb glycoproteins at alkaline conditions and the retained glycoproteins could be released at acidic conditions, hardly any IgG is recovered from the CPBA-Ni6PW9/SA hybrid even at pH 2.0. Besides, an elution efficiency of 36.8% is achieved at pH 10.0 in this study. IgG (pI 6.95) is positively charged at pH 2.0. Therefore, the electrostatic attraction between protein–surface (CPBA-Ni6PW9/SA) makes the elution of IgG from CPBA-Ni6PW9/SA hybrid is difficult. At pH 10.0, the electrostatic repulsion weakens the adsorption of IgG onto the CPBA-Ni6PW9/SA hybrid. Considering the strong hydrophilic interaction between CPBA-Ni6PW9/SA hybrid and IgG, Tris-HCl buffer is selected as the eluent. Tris contains a tripodal alcohol ligand and exhibits a stronger hydrophilic interaction with CPBA-Ni6PW9/SA
than IgG, which releases competitively the adsorbed IgG from the surface of CPBA-Ni6PW9/SA. At pH 10.0, by increasing the concentration of Tris-HCl buffer from 50 to 100 mmol L-1, the elution efficiency is increased from 71.3% to 82.1%. Therefore, in the future studies, 100 mmol L-1 of Tris-HCl (pH 10.0) is chosen as the eluent for recovering the retained glycoproteins from the CPBA-Ni6PW9/SA hybrid.
Figure 8. The recoveries of the adsorbed IgG from CPBA-Ni6PW9/SA hybrid with various eluents. Protein solution: 100 µg mL-1, 1.0 mL. CPBA-Ni6PW9/SA: 1.0 mg. Adsorption time: 30 min. BR buffer: 40 mmol L-1. Tris-HCl buffer: 50 and 100 mmol L-1. The volume of the stripping reagent: 1.0 mL. Stripping time: 30 min 3.6 Enrichment of glycoproteins from human serum with CPBA-Ni6PW9/SA The practicability of CPBA-Ni6PW9/SA hybrid for the adsorption of glycoproteins is carried out by isolating IgG from human serum. SDS polyacrylamide gel electrophoresis (SDS-PAGE) assay results are shown in Figure 9. Ten protein bands of the diluted human serum sample are identified which are mainly corresponding to HSA (66.4 kDa), IgG heavy chain (50 kDa), and IgG light chain (25 kDa) (lane 2). After recovering the proteins with Tris-HCl buffer solution, only the bands of IgG (lane 4) are observed. It is well consistent with the bands of IgG standard solution (lane 5). Image J software is used to analyze the intensity of the serum albumin at lane 2 and lane 4 [53]. It can be obtained by calculation that approximately 92% of the high abundance non-glycoprotein, i.e., serum albumin, is
removed during the adsorption process, and IgG is selectively separated from the human serum.
Figure 9. SDS-PAGE analysis of human serum before and after treatment with CPBA-Ni6PW9/SA. Lane 1: protein marker (kDa), lane 2: serum sample after 100-fold dilution without pretreatment by CPBA-Ni6PW9/SA, lane 3: 100-fold diluted serum sample after treating with CPBA-Ni6PW9/SA, lane 4: protein recovered from CPBA-Ni6PW9/SA, lane 5: IgG standard solution (150 µg mL-1). Moreover, LC-MS/MS is further adopted to evaluate the practicality of CPBA-Ni6PW9/SA hybrid in the isolation of glycoproteins. CPBA-Ni6PW9/SA hybrid is used to enrich glycoproteins from human serum under neutral environment and the absorbed proteins are recovered as described in the above sections. Through trypsin digestion and PNGase F deglycosylation, the obtained peptides are analyzed by LC-MS/MS. To ensure high confidence identifications, a minimum of two unique peptides per protein is adopted to filter proteins. There are 81 glycoproteins identified after treatment with the CPBA-Ni6PW9/SA hybrid, providing a recognition selectivity of 79.4%. The identified glycoproteins data are recorded in Table S1, suggesting that the CPBA-Ni6PW9/SA hybrid exhibits a favorable affinity to glycoproteins.
4. Conclusions A novel hybrid material for enrichment of glycoproteins is prepared by modifying NiII-substituted lacunary Keggin-type polyoxometalate with sodium
alginate via a one-pot hydrothermal approach, and subsequently functionalized with 4-carboxyphenylboronic acid through amide reaction. The obtained CPBA-Ni6PW9/SA hybrid significantly decreases the pKa value of CPBA to 6.5-7.0, attributing to the B-O coordination of B atom in CPBA and abundant oxygen atoms of Ni6PW9. Thus high adsorption selectivity towards glycoproteins is realized at mild environment of pH 7.0. This adsorption behavior of CPBA-Ni6PW9/SA is not only attributed to the boronate affinity interaction, but also due to the hydrophilic interaction. The present study offers a novel strategy for the reduction of the pKa value of boronic acid moiety, which is able to enrich glycoproteins from complex biological samples under neutral conditions.
5. Acknowledgements This work is financially supported by the Natural Science Foundation of China (21974018, 21727811, 21575020). Fundamental Research Funds for the Central Universities (N170505002, N170504017, N170507001).
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Highlights 1. The pKa value of 4-carboxyphenylboronic acidis successfully decreased via modification with a nickel-substituted polyoxometalate. 2. The well-designed hybrid is suitable for the selective adsorption of glycoproteins in neutral environment (pH 7.0). 3. The novel hybrid possesses excellent adsorption behavior toward glycoproteins based on boronate affinity interaction and hydrophilic interaction. 4. Highly specific enrichment of glycoproteins from human serum is achieved with the CPBA-Ni6PW9/SA hybrid.
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: