Article pubs.acs.org/Biomac
Preparation of Functionalized Alkynyl Magnetic Microspheres for the Selective Enrichment of Cell Glycoproteins Based on Click Chemistry Qingxin Cui,†,‡ Yuanyuan Hou,†,‡ Jie Hou,† Pengwei Pan,† Lu-Yuan Li,†,‡ Gang Bai,*,†,‡ and Guoan Luo†,‡,§ †
College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, People’s Republic of China ‡ Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People’s Republic of China § Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China S Supporting Information *
ABSTRACT: Functionalized alkynyl polyvinyl alcohol magnetic microspheres (PVA MMs) were developed for the specific enrichment of sialic acid-rich glycoproteins by click chemistry. The capture capability for proteins was evaluated through a novel dual-labeled bovine serum albumin (BSA) that utilizes fluorescence resonance energy transfer (FRET). The PVA MM parameters, including the size and coverage of functionalized groups, were optimized by response surface methodology. The optimal parameters obtained were 1.25− 6.31 μm in size and 48.53−73.05% in coverage. Then, the optimal PVA MMs were synthesized, and the morphology and surface chemical properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). To capture glycoproteins from the cell surface, a bioorthogonal chemical method was applied to metabolically label them with an azide group. The functionalized alkynyl PVA MMs showed a high specificity and strong binding capability for glycoproteins through a [3 + 2] cycloaddition reaction. The results indicated that the functionalized alkynyl PVA MMs could be applied to the enrichment of cell glycoproteins, and the merits of the MMs suggested an attractive and potential way to facilitate glycoprotein research.
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INTRODUCTION Over the past decade, magnetic microspheres (MMs) have received increasing attention because of their unique physical and chemical properties as well as their many potential applications in various fields such as drug delivery and cell separation.1−4 The application of MMs to biological research has increased quickly5−8 because of their strong magnetic properties that facilitate the isolation of MM−target protein complexes from the multifarious sample. Unlike conventional separation techniques, the separation process based on magnetic supports can be performed directly in complex samples without any pretreatment.9,10 The functional groups on the surface of MMs are important for bioapplications and need to be suitable for conjugation with biomolecules, such as enzymes,11,12 antibodies,13,14 and nucleotides.15,16 However, the parameters of MMs, including the size and coverage of functionalized groups, with properties for specific glycoprotein enrichment remain unknown. Glycosylation is an important post-translational modification that occurs in 50% of eukaryotic proteins.17 These modifications play a crucial role in various biological events, such as cell recognition, intracellular signaling, cell adhesion, and cell−cell interaction. 18 Changes in glycosylation are often a hallmark of disease states.19 Sialic acid, transferred from mannose, is typically the outermost © 2012 American Chemical Society
monosaccharide on glycan chains of glycolipids and glycoproteins, has an indispensable function in normal cells, and plays a central role in physiological and pathological processes. The levels of glycoproteins modified by sialic acid were strongly associated with the development of cancer.20 Therefore, it is imperative to investigate the expression and enrichment of sialic acid-rich glycoproteins to better understand cancer progression. Many methods for assaying glycoprotein enrichment that target different fundamental structures or subunits have been reported. The hydrazide chemistry method was generally carried out by oxidizing the sugar residues, followed by attachment to a hydrazide resin to capture the glycosylated peptides.21 Lectins were found to have affinity for glycolipids and glycoproteins through interactions with various carbohydrate moieties. This characteristic has been used in chromatography for purifying glycoproteins.22,23 Although these methods are the most widely used approaches to enrich discrete glycoproteins, they can cause significant perturbations to a protein’s structure and have no direct extension to the secondary metabolites.24 In recent years, an alternative tool for Received: September 20, 2012 Revised: November 15, 2012 Published: December 5, 2012 124
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Figure 1. (A) Fluorescence labeling and enrichment scheme of BSA. (B) Fluorescence emission spectra diagram for this study.
biocompatibility. Then, the functionalized alkynyl PVA MMs were prepared by a suspension embedding procedure. The enrichment capability of functionalized alkynyl PVA MMs were evaluated by a dual-labeled bovine serum albumin (BSA) tag based on click chemistry. Response surface methodology was used to optimize the significant factors of MMs, and the parameters of optimal MMs conditions were determined. Then, the alkynyl PVA MMs were synthesized and characterized with transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Furthermore, functionalized MMs were applied to capture the metabolically labeled glycoproteins of A549 cells. The novel alkynyl PVA MMs showed significant glycoprotein capture and release capabilities. Therefore, we have provided a method for the simple enrichment of sialic acid-rich glycoproteins from the cell surface.
tagging biomolecules has emerged from the chemical biology community, the bioorthogonal chemical reporter.24 In a prototypical experiment, a unique detectable chemical motif, often as small as a single functional group, is incorporated into the target biomolecule using the cell’s own biosynthetic machinery.25 The chemical reporter is then covalently modified in a highly selective fashion with an exogenously delivered probe.26 In glycoprotein analysis, mannose is modified with a reactive chemical group. Non-natural mannose can be utilized and metabolized as a form of sialic acid using the cell’s metabolic system. Then, the chemical tags can be detected for isolation or visualization.27 Azido was found to be an ideal group that is reactive with alkynes, meets these criteria, and is considered to be a nonperturbing and highly selective reaction.25,28,29 The exemplary copper(I) catalyzed azide− alkyne cycloaddition30 has excellent attributes in high efficiency and high selectivity. Protein separation based on click chemistry will enlarge the bioapplication and increase specificity.31−33 In the present work, to establish MMs that are suitable for glycoprotein capture, polyvinyl alcohol (PVA) was used to prepare polymer MMs because of their hydrophilicity and
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EXPERIMENT SECTION
Reagents and Materials. Ferric chlorides, 6-hydrate and ferrous chloride tetrahydrate were purchased from Sigma-Aldrich (St. Louis, U.S.A.). PVA was purchased from Beijing Chemical Reagents 125
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Figure 2. (A) Contour plot of interaction between size and coverage of functional groups; (B) response surface plot. (ENEN) were synthesized according to previous studies.29,36 In this experiment, for a given pH of 9.0, BSA (3.4 mg, 5 × 10−8 mol) was dissolved in 5 mL of sodium bicarbonate buffer solution (0.1 mol/L, pH 9.0). Then, the NHS-N3 (0.13 mg, 5 × 10−7 mol) and NHSfluorescein (0.24 mg, 5 × 10−7 mol) were added into the above solution. This system was treated with ultrasound for 2 h at room temperature. Then, centrifugation was used to obtain the supernatant. The supernatant was added to a 50 mL ultrafiltration tube and washed with 20 mL of phosphate buffered saline (PBS, pH 7.4) several times until fluorescein could no longer be detected in the filtrate. Finally, the N3-BSA-fluorescein tag solution was diluted to 1 mL and supplied with catalyst (0.2 mmol/L Tris-triazoleamine, 1.0 mmol/L CuSO4, and 2.0 mmol/L sodium ascorbate). Then, ENEN (0.013 mg, 10−8 mol) was added to the BSA solution, and the resulting suspension was shaken at 4 °C for 1 h. The synthesis and click reaction principle are described in Figure 2A. Measurement of the Capturing Ability of Alkynyl MMs by Dual-Labeled BSA Tag. The N3−BSA-Fluorescein tag solution was diluted to 1 mg/mL and supplied with catalyst. Then, 1 mL functionalized MMs of different sizes and coverages (10 mg of beads/mL, prewashed four times with ice-cold hypotonic buffer before use) were added to 10 mL of N3−BSA-Fluorescein tag solution, and the resulting suspension was shaken at 4 °C for 1 h. Then, the beads were collected by magnetic separation and washed three times with 1 mL of precooled PBS. The captured BSA-fluorescein tag was released with 100 mmol/L dithiothreitol (DTT) solution for 2 h. Detection of the N3-BSA-fluorescein tag, ENEN-BSA-fluorescein tag, and released BSA-fluorescein tag was carried out by a full wavelength fluorescence scanning luminoscope (Tecan Safire 2 type, Switzerland). The excitation wavelength was 365 nm, and the emission wavelength was from 400 to 700 nm. Then, the maximum emission wavelength was determined, and the released BSA-fluorescein tag was detected by the luminoscope. Characterization of Functionalized Alkynyl MMs. The morphology and structure of the functionalized alkynyl MMs were studied by using a scanning electron microscope (FEI SIRION 200 with 200 kV accelerated voltage, U.S.A.) and a transmission electron microscope (JEOL JEM-2010, U.S.A.). The particle hydrodynamic size was measured by using a Counter laser size analyzer (Beckman Coulter, U.S.A.). Magnetic measurement was performed using a superconducting quantum interference device (SQUID) Quantum Design (San Diego, U.S.A.) MPMS7 magnetometer. The PEI coating of the magnetic nanoparticles was checked using a Perkin-Elmer (Norwalk, U.S.A.) Spectrum GX Fourier transformation infrared (FTIR) spectrometer (Nicolet NEXUS 670) using KBr pellets. Metabolic Labeling of Cell Glycoproteins. Sialic acid-rich glycoproteins play an important role in the occurrence and development of tumors, so azide-labeled mannose, which can be metabolized into sialic acid, was chosen as the chemical reporter.25 In this study, peracetylated N-azidoacetylmannosamine (Ac4ManNAz) and biotin-SS-alkyne were synthesized according to our previous
Company (Beijing, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Alfa Aesar (Massachusetts, U.S.A.). NHS-fluorescein and avidinCy3 were purchased from Pierce (Rockford, U.S.A.). The 1640 medium and newborn calf serum were from Gibco BRL Life Technologies (Rockville, U.S.A.). A549 cells were obtained from American Type Culture Collection (Rockville, U.S.A.). All other chemicals and solvents used were analytical grade. The water used throughout this work was reagent-grade water produced by the Milli-Q SP Ultra-Pure Water Purification System of Nihon Millipore Ltd. (Tokyo, Japan). Synthesis of Functionalized Alkynyl PVA MMs. Fe 3 O4 magnetite nanoparticles were made according to previously reported methods of coprecipitation.10,34 PVA MMs were prepared by a suspension embedding procedure according to the aforementioned method.35 Briefly, Fe3O4 magnetite nanoparticles were added to a 5% (w/v) PVA (degree of polymerization: 1750 ± 50) aqueous solution. The mixture was well dispersed in an ultrasonic bath. In a reactor equipped with a stirrer, an organic mixture of petrol (200#), tetrachloromethane, and span-80 (1/1/0.08, v/v/v, respectively) was stirred for 30 min at room temperature. The mixture of organic solution and PVA solution was well dispersed using a homogenizer (FA25, Fluco, England), added to the reactor, and dispersed at 1000− 24000 rpm. Subsequently, 1 mol/L HCl was added slowly to the mixture. Immediately after starting the suspension process, a 25% glutardialdehyde solution was added and the final concentration was 0.5%. Suspension cross-linking proceeded for 1 min, the temperature raised slowly to 70 °C, and the solution was stirred for 2 h. After cooling to room temperature, PVA MMs of different sizes were magnetically isolated, washed with ethanol and water, and then stored in distilled water at 4 °C. PVA MMs (3.9 g, 22.2 mmol), 4 mL of 50% NaOH aqueous solution (51 mmol), and tetrabutylammonium bromide (709 mg, 2.2 mmol) were successively added at room temperature, and 4 mL of hexane and (R)-(−)-epichlorohydrin were then added in 1−10 μL drops,8 and the resulting suspension was stirred for 12 h at 60 °C. After cooling to room temperature, the solution was washed with ether and water. The combined organic phases were epoxy MMs with different grafting densities. Finally, 10 mL of 10% ammonia in water was added to the epoxy MMs, and the reaction system was stirred for 4 h at room temperature. After magnetic separation, the amino MMs had been synthesized. The synthesis of 2,5-dioxopyrrolidin-1-yl 2-[(2-oxo-2-(prop-2ynylamino) ethyl) disulfanyl] acetate (DPYED) is depicted in the Supporting Information (Scheme 1). DPYED (316 mg, 1 mmol) was dissolved in 0.2 mL of borate buffer (0.2 mol/L pH 8.0), and 1 mL amino MMs was added. The mixture was stirred at room temperature for 4 h. The functionalized alkynyl MMs were collected by magnetic separation and washed three times with water. The general strategy for synthesis of functionalized alkynyl MMs was shown in Figure 1A. Synthesis of N3-BSA-Fluorescein Tag. 2,5-Dioxopyrrolidin-1-yl6-azidohexanoate (NHS-N3) and 4-ethynyl-N-ethyl-1,8-naphthalimide 126
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study.37 A549 cell line, which is derived from human bronchial epithelial cells, were cultured using 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 IU/mL penicillin, 100 IU/mL streptomycin, and 0.2 mmol/L Ac4ManNAz for 48 h. A549 cells were washed with precooled phosphate-buffered saline (PBS, 0.1 mol/L, pH 7.4) and treated with a 100 μL reaction solution (0.2 mmol/L biotin-SS-alkyne, 0.2 mmol/L Tris-triazoleamine, 1.0 mmol/L CuSO4, and 2.0 mmol/L sodium ascorbate in precooled PBS). After 1 h incubation, the cells were washed with PBS, fixed using 4% paraformaldehyde, and permeabilized with 1% Triton X100 for 30 min. Then, the cells were labeled with 0.2 mM streptavidin-Cy3. After fluorescent labeling, 100 mmol/L dithiothreitol (DTT) was added to determine whether the Cy3 could be released. All fluorescence images were obtained with a confocal microscope (TCS SP5, Leica, Germany). The excitation wavelength was 543 nm, and the emission wavelength was 570 nm. Capture of Glycoproteins from Cell Surface. A549 cells were cultured with Ac4ManNAz in culture dishes (10 cm) as described above. Cells were scraped into precooled PBS (0.01 M, pH = 7.4) and incubated on ice for 15 min. The cells were lysed by sonication. After centrifugation at 12000 rpm for 10 min at 4 °C, the supernatant was collected and the protein content was quantified with a BCA Protein Assay Kit (Thermo, U.S.A.). The supernatant was diluted to 1 mg/mL and supplied with catalyst. Then, 1 mL of functionalized alkynyl MMs (10 mg of beads/mL, prewashed four times with ice-cold hypotonic buffer before use) was added to 10 mL of lysate, and the resulting suspension was shaken at 4 °C for 1 h. Then, the beads were collected by magnetic separation and washed with 1 mL of precooled PBS. The glycoproteins were released with 100 mmol/L DTT or 0.1 mol/L PBS solution, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then silver staining was performed.
during the enrichment of target proteins:37 the coverage of functional groups and the size of the MMs. Therefore, the two effective parameters were systematically evaluated. Using a 5% emulsion concentration, five preparations of MMs with various sizes were produced by controlling the dispersal rate from 3000 to 22000 rpm. The MM diameters ranged from 300 nm to 50 μm. The particle size distribution is consistent with “the smallest critical size theory” previously reported in the literature.35 The concentration of functional groups was determined by the number of epoxy groups according to the literature.8 According to the saturation of functional groups of different sizes, we can calculate the relative coverage of each MM. To prepare the optimal MMs to capture the maximum amount of protein, central composite design (CCD)38 was used to design the experiment. The domain of variation for each factor was determined based on knowledge of the system and acquired from initial experimental trials. A total of 13 experiments were carried out; size (A) and the coverage (B) variables as well as their domains are shown in Table 1. In this design, experiments Table 1. Response Surface Experiments Design and Response Values
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RESULTS AND DISCUSSION Detection of Fluorescently Labeled BSA Tag. To evaluate the enrichment capacity of alkynyl MMs for protein capture based on click chemistry, N3-BSA-fluorescein tags were synthesized using NHS-fluorescein and NHS-N3, which reacted with primary amino groups of BSA to form stable amide bonds. As shown in Figure 1A, the N3-BSA-fluorescein tag not only conjugated with ENEN through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction and emitted a blue fluorescence, but also could be detected by fluorescein with green fluorescence. The excitation and emission wavelengths of the triazole group in the labeled BSA tag were verified at 365 and 465 nm and at 488 and 518 nm for the fluorescein group, respectively. Fortunately, the fluorescence resonance energy transfer (FRET) could be observed in the dual-labeled BSA tag: the triazole could be a donor and the fluorescein an acceptor. The excitation wavelength was determined to be 365 nm, and the emission wavelength was set at 518 nm (Figure 1B). The FRET of released BSA-fluorescein tag was also detected at the same wavelength, but the fluorescence intensity was weaker than the ENEN-BSA-fluorescein tag. The FRET of the N3-BSAfluorescein tag could not occur because it only contained fluorescein. Therefore, the N3-BSA-fluorescein tag could be used to evaluate the protein capture capabilities of alkynyl MMs based on click chemistry by FRET specifically. Optimization of the Alkynyl MM Parameters Based on Response Surface Methodology. The alkynyl groups on the end of the MMs grafting provide recognition sites for the azidolabeled protein, and the disulfide bond in the middle of the grafting provides a reversible cleavage site in the BSAfluorescein tag captured MMs. The enrichment capability of the MMs was evaluated by detecting the fluorescence intensity of the released BSA-fluorescein tag. Previous studies have shown that two parameters of the MMs have to be considered
run
A-size (log−1) (μm)
B-coverage (%)
fluorescence intensity
1 2 3 4 5 6 7 8 9 10 11 12 13
1.62 0.56 0.56 0.56 0.56 0.56 −0.94 1.62 0.56 2.06 −0.50 0.56 −0.50
100.00 100.00 60.00 3.43 60.00 60.00 60.00 20.00 60.00 60.00 100.00 60.00 20.00
12.13 47.37 75.92 36.45 77.31 74.68 19.20 21.84 75.66 13.71 15.96 72.87 28.74
required for optimizing two factors are shown in Table 2. This design includes the possibility of evaluating cross-effects between variables without the need to perform additional experiments. The software Design-Expert 7.0 (Stat-Ease, Inc.) was utilized to analyze the results, and the secondary linear Table 2. ANOVA Results of Regression Analysis item model A-size B-coverage AB A2 B2 R2 optimal parameters scale predicted value (max) actual parameters actual value (max) 127
sum of squares
DF
mean square
8619.16 5 1723.83 42.71 1 42.71 5.19 1 5.19 4.00 1 4.00 7007.09 1 7007.09 2508.00 1 2508.00 0.9587 size: 1.25−6.31 μm; coverage:
F
P
31.76 0.79 0.096 0.074 129.10 46.21
0.0001 0.4045 0.7661 0.7939
