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Prepare concanavalin A-chelating magnetic nanoparticles for selective enrichment of glycoproteins Liping Dong, Shun Feng, Shanshan Li, Peipei Song, and Jide Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01184 • Publication Date (Web): 11 Jun 2015 Downloaded from http://pubs.acs.org on June 22, 2015
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Prepare concanavalin A-chelating magnetic nanoparticles for selective enrichment of glycoproteins Liping Dong, Shun Feng*, Shanshan Li, Peipei Song, Jide Wang Key Laboratory of Oil Gas and Fine Chemicals, Ministry of Education and Xinjiang Uyghur Autonomous Region, College of Chemistry and Chemical Engineering, Xinjiang University, Urumqi 830046, China Corresponding Author * Phone: +86-991-8582087. Fax: +86-991-8582087. Email:
[email protected] (Shun Feng).
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Prepare concanavalin A-chelating magnetic nanoparticles for selective enrichment of glycoproteins Liping Dong, Shun Feng*, Shanshan Li, Peipei Song, Jide Wang Key Laboratory of Oil Gas and Fine Chemicals, Ministry of Education and Xinjiang Uyghur Autonomous Region, College of Chemistry and Chemical Engineering, Xinjiang University, Urumqi 830046, China ABSTRACT: In this work, a soft and non-destructive approach was developed to prepare concanavalin A-chelating magnetic nanoparticles (Con A-MNPs) for selective enrichment of glycoproteins. Ethylenediamine tetraacetic acid-modified-MNPs (EDTAMNPs) were prepared by one-pot chemical co-precipitation method firstly, and then Cu(II) cations were used as bridge groups to immobilize Con A on EDTA-MNPs. The as-prepared absorbents with a mean diameter of 15 nm showed a strong magnetic response to an externally applied magnetic field. The results of thermogravimetric analysis showed the content of immobilized Con A was up to 28 wt%. To glycoprotein ovalbumin, the maximum capacity and equilibrium constant were 72.41 mg/g and 0.6035 L/mg, respectively. The as-prepared nano-composites exhibited a remarkable selectivity for glycoproteins, and can enrich glycoproteins specifically from a mixture of glycoprotein and non-glycoprotein even at a molar ratio of 1:600. It was also successfully applied into enriching glycoproteins from real egg white sample. We expect that our finding will serve as a helpful template for others to design new adsorbents for enriching glycoproteins.
Glycosylation of proteins is the most ubiquitous posttranslational modification observed in eukaryotic organisms, plays crucial roles in various physiological processes.1,2 Aberrant glycosylation has been recognized to be associated with several disease states such as cancer, inflammatory diseases, and congenital disorders, etc.3-6 Given the major roles glycoproteins play in cellular functions, numerous efforts have been focused on improving the efficiency of glycoprotein analysis. However, it is difficult to analyze due to the presence of other high abundant proteins and wide dynamic range in complex biological samples. Therefore, efficient isolation and enrichment of glycoproteins are indispensable in order to obtain an in-depth understanding of glycoproteins. Various analytical tools have been developed for enrichment of glycoproteins, including hydrazide chemistry3-5, boronic acid chemistry6,7, hydrophilic interaction liquid chromatography8-11 and lectin-based affinity chromatography12-15. Among which, lectin-based affinity chromatography is most widely used to enrich glycoproteins from complex biosamples.16-18 Concanavalin A (Con A), one lectin binding specifically with α-D-mannosyl and α-D-glucosyl residues in glycoproteins,16,19 is popularly applied into N-glycoproteins enrichment by immobilized on a certain support based on different mechanism, such as physical adsorption17, chemical binding18, as well as affinity interaction20,21. In our group, one Con A-Cu(II)-iminodiacetic acid monolithic capillary column was successfully prepared to enrich glycoproteins based on chelating interaction.22 The results showed the column possessed many merits, such as high capacity (double than those based on covalently bound methods), non-destructive to lectin, and most importantly, ammonium hydroxide was used to elute the captured glycoprotein instead of the sugar-based elution buffer, which can avoid loss of glycoprotein yield and potential sample contamination caused by either multi-step or extensive wash cycles. Con A chelating materials provides a rapid and efficient alternative for its time-saving, easy operation, few washing steps, and high yield. In last decade, magnetic separation draws much attention due to its unique advantages such as durable magnetic susceptibility, high surface-to-volume ratio. And separations can be performed with an external magnetic field without any addi-
tional centrifugation or filtration procedures.23 It also have been used as substrate to enrich glycoproteins through grafted hydrophilic groups, which provided convenient and efficient enrichment approaches for glycoproteins.24-26 But to the best of our knowledge, there was no report about the preparation of lectin-based magnetic nanoparticles (MNPs) through chelating interaction. In this work, ethylenediamine tetraacetic acid (EDTA)-functionalized MNPs were prepared by one-pot chemical co-precipitation method, and then a soft and nondestructive approach was used to immobilize Con A through chelating interaction using copper(II) as bridge group, and resulted a Con A chelating MNPs with a stable sandwich structure: Con A-Cu(II)-EDTA. The facile immobilization process of Con A onto magnetic nanoparticles is shown in Scheme 1. The capacity, selectivity and specificity of Con ACu(II)-EDTA-MNPs (Con A-MNPs) were investigated using standard protein mixtures and real egg white samples. The results testified that the as-prepared Con A-MNPs exhibited significant selectivity and specificity to glycoproteins, and showed a high potential for large-scale glycoproteomics research in complicated biological samples.
Scheme 1. The synthesis of EDTA-functionalized magnetic nanoparticles and Con A-immobilized procedure.
EXPERIMENTAL SECTION Materials and reagents Con A from canavalia ensiformis and lysozyme (lyz) from chicken egg white were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Ovalbumin (OB) from chicken egg white, coomassie brilliant blue R-250, acrylamide, tris (hydroxymethyl) aminomethane (Tris), sodium dodecyl sulfate
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(SDS), N,N,N’,N’-Tetramethylethy lenediamine (TEMED) and ammonium persulfate were obtained from Aladdin Reagents (Shanghai) Co., Ltd. (Shanghai, China). Unstained protein molecular weight marker (14.4-116 kDa) was purchased from Thermo Fisher Scientific, Inc (Chengdu, China). Iron(III) chloride hexahydrate (FeCl3.6H2O), ferrous sulfate heptahydrate (FeSO4.7H2O), ammonium hydroxide (25%), ethylenediamine tetraacetic acid (EDTA) were purchased from Tianjin Chemical Reagent Co., Ltd (Tianjin, China). All other chemicals were the guaranteed or analytic grade reagents commercially available and used without further purification. Binding buffer: 20 mM Tris, 0.15 M NaCl, 1 mM MnCl2, 1 mM CaCl2, pH 7.4. SDS-PAGE sample buffer: 4 mL distilled water, 1 mL 0.5 M pH 6.8 Tris-HCl, 0.8 mL glycerin, 1.6 mL 10% SDS, 0.4 mL 2-mercaptoethanol, 0.2 mL 0.1% bromophenol blue. 5× Running buffer: 15 g of Tris, 72 g of glycine and 5 g of SDS in 1000 mL of water. Solution A: a mixture of binding buffer with equal volume of SDS-PAGE sample buffer. Preparation of Con A-MNPs The EDTA-Fe3O4 magnetic nanoparticles (EDTA-MNPs) were firstly prepared using a one-pot chemical co-precipitation method with few modification.27 Briefly, 3.05 g of FeCl36H2O and 2.1 g of FeSO47H2O were dissolved in 50 mL distilled water and heated to 90°C, respectively. Then the two solutions, 5 mL of ammonium hydroxide (25%) and 0.615 g of EDTA dissolved in 25 mL of ammonium hydroxide (1.5%) were mixed rapidly and sequentially. The resulted mixture was stirred at 90°C for 30 min and then cooled to room temperature. The black precipitate was collected by a permanent magnet and washed to neutral pH with distilled water. The obtained black precipitate was collected and ready for use. Immobilization of copper ions was accomplished by adding 1.5 mg EDTA-MNPs in an 1 mL 0.5 M CuSO4 (pH 5.0) aqueous solution for 1 h under gentle stirring to produce Cu(II)EDTA-MNPs (Cu-MNPs). The resulted products was collected by a permanent magnet, and washed three times with distilled water (pH 5.0). For the binding of Con A, the Cu-MNPs were washed three times with 500 µL binding buffer at first, and then mixed with 1 mL of Con A solution (1 mg/mL in binding buffer) under gentle shaking for 1 h at room temperature. The resulting Con A-MNPs were collected magnetically, and washed three times with binding buffer for further using. Characterization Transmission electron microscopy (TEM) image was obtained by JEM-2010 Transmission Electron Microscope (JEOL, Tokyo, Japan). Fourier transform infrared (FTIR) spectra were recorded on Vertex 70 FTIR spectrometer (Bruker, Saarbrücken, Germany). Thermogravimetric analysis (TGA) were performed on SDT Q600 (TA Instruments, New Castle, US) under air flow. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained from Bio-Rad laboratories (Bio-Rad, Hercules, USA). The data of adsorption were obtained by using a T6 New Century UV/Vis spectrophotometer (Pgeneral, Beijing, China). Adsorption of standard proteins mixtures by Con AMNPs An amount of 2 mg Con A-MNPs was dispersed in 2 mL of OB solution (25-200 mg/L, dissolved in binding buffer). After
incubated for a certain time at room temperature with gentle shaking, the Con A-MNPs were isolated magnetically, and the content of protein in the supernatant was determined using a UV/Vis spectrophotometer at a wavelength of 280 nm. Selective adsorption experiments were performed using mixtures of standard glycoprotein OB and non-glycoprotein Lyz with different molar ratios of 1:300 and 1:600. An amount of 2 mg Con A-MNPs was dispersed in 2 mL of mixtures solution with an initial amount of OB 50 µg. After incubation, the Con A-MNPs were isolated magnetically and washed five times with 500 µL binding buffer to remove non-specific bound protein, all washing buffers were collected and mixed together. Subsequently, the adsorbents were immersed in 500 µL elution buffer (1 M ammonium hydroxide) to elute the captured glycoproteins. Finally, washing buffer or the eluted fractions were dried in a vacuum centrifuge for further SDSPAGE analyses. Glycoproteins enrichment from real egg white samples by Con A-MNPs For glycoprotein enrichment from real egg white sample, a mount of 6 mg Con A-MNPs were incubated in 300 µL of 100-fold or 500-fold dilutions of egg white samples in binding buffer under gentle shaking for 30 min. After incubation, the Con A-MNPs were isolated magnetically and washed five times with 500 µL binding buffer to remove non-specific bound protein. Subsequently, the adsorbents were immersed in 500 µL elution buffer to elute the captured glycoproteins. Finally, the supernatant and the eluted fractions were dried in a vacuum centrifuge for further SDS-PAGE analyses. SDS-PAGE To standard proteins mixtures, the dried washing fractions were redissolved in 500 µL solution A. The dried eluted fractions were redissolved in 50 µL solution A. In turns of real egg white samples, all the dried fractions were redissolved in 300 µL solution A. And then the samples were analyzed by SDSPAGE after heat at 95oC in a water bath for 4 min. The loading amount is 3-5 µL based on the concentration of the samples. The SDS-PAGE of the samples were conducted on the 12% SDS-polyacrylamide gel in running buffer, electrophoresed at 28 mA for 45 min, then stained with coomassie brilliant blue R-250 for 20 min.
RESULTS AND DISCUSSION Characterization of Con A-MNPs Typical TEM images of EDTA-MNPs and Con A-MNPs were shown in Fig. 1 (a). It can be seen that either EDTA-MNPs or Con A-MNPs were discrete with a mean diameter of 15 nm, which indicated that the binding process did not significantly change the particle size, and Con A-MNPs can keep the stability of nanodispersions. Meanwhile, the Con A-MNPs showed a rapid response to an externally applied magnetic field and the separation can be completed within 15 s (Fig. 1 (b)). FTIR spectroscopy was used to demonstrate the binding of Cu2+ and Con A successfully. As shown in Fig. 1 (c), the peak around at 570 cm-1 in curves A-C was ascribed to the Fe-O band, and the peaks at 3385 cm-1 and 3365 cm-1 in curves A and B were assigned to the -OH vibrations of EDTA. Compared with cure A, The IR adsorption peak of -OH in curve B shifted to lower wave number, revealing that Cu2+ was bound on the surface of EDTA-MNPs. Two main characteristic peaks
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similar conclusion. Such a fast adsorption rate was mainly due to the soft and non-destructive immobilized approach, which can keep the bioactivity of Con A. 60
q (mg/g)
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Figure 2. Adsorption kinetic curves of Con A-MNPs, and with an initial OB concentration of 100 µg/mL in binding buffer at pH 7.4. 80
A UV-vis spectrophotometric method was firstly performed to determine the amount of the bound Con A. A volume of 500 µL elution buffer was used to elute the bound Con A from 2 mg Con A-MNPs, and then the quantity of Con A in eluate was calculated based on standard curve method at 280 nm. It was found the average amount of bound Con A in the Con AMNPs was as large as 30.16 wt% (n=3). TGA was further executed to quantitatively estimate the related composition of Con A-MNPs. As shown in Fig. 1 (d), the TGA curves of A and B showed two main weight loss steps. The first one over the temperature range from 30 to 180oC was due to the loss of residual water in the samples. The second one (mainly at 180400oC) was due to the decomposition of EDTA. Different from curve A, the weight percentage of curve B is a little higher than that of curve A, which was caused by the conversion of Cu2+ to CuO due to the high temperature treatment in air. The curve C showed that the weight loss over 200oC should be resulted mainly by the decomposition of Con A. Based on TG result, it can be calculated that the content of Con A is about 28 wt% in Con A-MNPs, which was a few less than the result derived from UV-vis method. It was reasonable, Cu2+ in eluate may interfere with the determination of Con A in UV-vis spectrophotometric method. The results of either UV-vis or TG method showed the immobilized amounts of Con A were around 30 wt%, which was much higher than previous reports, in which it was only in the range of 1-10 wt% whatever based on chemical reaction or hydrophilic interaction29-32. The bigger amount of immobilized Con A was, the higher adsorption capacity could be reached. Adsorption of standard glycoprotein Fig. 2 presented the adsorption kinetic curves of Con A-MNPs to OB. It can be seen that the adsorption equilibrium could be reached in 10 min with an initial OB concentration of 100 µg/mL, which was much more quickly than those of commercial lectin based-resins. The work by Lai et al.30 also got the
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Figure 1. Characterization of Con A-MNPs. (a) TEM image of A, EDTA-MNPs and B, Con A-MNPs; (b) Magnetic response of Con A-MNPs to a magnet; (c) FTIR spectra of A, EDTA-MNPs; B, Cu-MNPs and C, Con A-MNPs; (d) TGA curve for A, EDTAMNPs; B, Cu-MNPs and C, Con A-MNPs.
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Figure 3. Adsorption isotherm for the adsorption of OB by Con A-MNPs in binding buffer (pH 7.4). The inset illustrates the linear dependence of Ce/qe on Ce.
The adsorption isotherm for the adsorption of OB by Con A-MNPs was investigated in the binding buffer, and the adsorption equilibrium data were fitted with Langmuir isotherm equation: Ce Ce 1 = + qe qm q m K L where qe is the equilibrium adsorption capacity (mg/g), Ce is the equilibrium OB concentration in solution (mg/L), qm is the maximum capacity (mg/g), and KL is the Langmuir adsorption constant (L/mg). From Fig. 3, it can be seen that the adsorption capacity of the nano-composites increased gradually with increasing Ce of OB, and approached to a maximum after Ce reached at 25 mg/L. As indicated in the inset of Fig. 3, a good linear relationship between Ce/qe and Ce was obtained with a correlation coefficient of 0.9992. It revealed the adsorption of OB on Con A-MNPs obeyed the Langmuir adsorption isotherm. The values of qm and KL were calculated as 72.41 mg/g and 0.6035 L/mg, respectively. Selective glycoprotein enrichment by Con A-MNPs Mixtures of standard glycoprotein OB and non-glycoprotein Lyz with different molar ratios of 1:300 and 1:600 were used to investigate the selectivity and specificity of the nanocomposites. As shown in Fig. 4, while the mole ratio of OB:Lyz even
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reached at 1:600, it was found that Lyz (14.4 kDa) cannot be observed in the eluate (E3) and only existed in washing solution (W3), meanwhile OB (45 kDa) can be only observed in the eluate (E3). The result demonstrated the remarkable selectivity and specificity of the Con A-MNPs to glycoprotein. Four bands below 35 kDa were originated from the dissociation of Con A.22,33 Interestingly, glycoprotein OB was observed only in elution (E1 and E2) but not in washing solutions (W1 and W2), which meant both of EDTA-MNPs and Cu-MNPs also showed selectivity to glycoprotein in some degree due to the weak hydrophilic interaction between Nglycans of glycoproteins and the carboxyl or hydroxy of EDTA.34 But the non-specific adsorption of EDTA-MNPs and Cu-MNPs to proteins was still dominant caused by interactions between the large amount of functional groups in proteins (NH2 or COOH) and copper or iron ions on the surface of the particles. The results demonstrated that Con A was covered the whole surface of Con A-MNPs and avoided the nonspecificity adsorption of the nano-composites caused by EDTA, copper or iron ions.
coproteins were captured by Con A-MNPs, and resulted in no glycoproteins observed in S2. The results of SDS-PAGE further testified the high selectivity and specificity of Con AMNPs to glycoproteins. kDa 116.0
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Figure 5. SDS-PAGE analysis of egg white samples. M: marker; ×100: 100-fold dilution of egg white before treatment with Con A-MNPs; S1: 100-fold dilution of egg white after treatment with Con A-MNPs; E1: elution; ×500: 500-fold dilution of egg white before treatment with Con A-MNPs; S2: 500-fold dilution of egg white after treatment with Con A-MNPs; E2: elution. Injected amount of the samples: 4 µL.
CONCLUSIONS
Figure 4. SDS-PAGE results of the mixture of OB and Lyz with a molar ratio of 1:300 adsorbed by EDTA-MNPs and Cu-MNPs, and with a molar ratio of 1:600 adsorbed by Con A-MNPs. M: protein marker; W: washing solution; E: elution. Injected amount of W1, E1, W2 and E2: 5 µL; W3 and E3: 3 µL.
Enriching glycoproteins from real egg white samples In order to demonstrate the practical applications of the Con A-MNPs, it was used to enrich glycoproteins from a real sample fresh egg white. Firstly, 300 µL of 100-fold or 500-fold diluted egg white samples was treated with 6 mg Con AMNPs according to the procedure described in the experimental section, and then analyzed by SDS-PAGE. As shown in Fig. 5, the bands of glycoproteins, ovotransferrin (76.7 kDa), ovoinhibitor (49 kDa), and OB (46 kDa), and nonglycoprotein Lyz (14.4 kDa) appeared in both of 100-fold and 500-fold dilutions. After enriching with Con A-MNPs, only bands standing for three glycoproteins and Con A were observed in the elution (E1 and E2 in Fig. 5), meanwhile Lyz can be observed in supernatant. It should be pointed that the total amount of glycoproteins in 300 µL of 100-fold diluted sample was reached at about 2.1 mg, which was much more than the theoretic adsorption capacity of Con A-MNPs (about 430 µg), which resulted in glycoprotein bands also existed in S1. While the real egg sample was diluted to 500-fold, the total amount of glycoproteins was decreased to around 430 µg, which was equivalent to the theoretic adsorption capacity of Con AMNPs. After enrichment, it can be found that almost all gly-
A soft and non-destructive method was proposed to prepare Con A-chelating MNPs with a stable sandwiched-structure for the fast magnetic selective separation of glycoproteins. The asprepared Con A-MNPs showed many merits, such as large enrichment capacity (72.41 mg/g to OB), fast magnetic separation speed (15 s), and relative quick enrichment process. It should be pointed that it can specifically enrich glycoproteins from a mixture of glycoprotein and non-glycoprotein even at a molar ratio of 1:600, which testified the Con A-MNPs possess excellent selectivity and specificity to glycoproteins. Furthermore, the Con A-MNPs was successfully applied into the glycoprotein enriching from real egg white samples, showing great potential in the enriching low-abundance glycoproteins in real complex biological systems. It provides a novel procedure to prepare magnetic nano-adsorbents for glycoprotein enrichment.
AUTHOR INFORMATION Corresponding Author * Phone: +86-991-8582087. Fax: +86-991-8582087. Email:
[email protected] (Shun Feng).
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by National Natural Sciences Foundation of China (21075105 and 21165017).
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