Core–Corona Magnetic Nanospheres Functionalized with Zwitterionic

Article Cite This: Biomacromolecules XXXX, XXX, XXX−XXX

pubs.acs.org/Biomac

Core−Corona Magnetic Nanospheres Functionalized with Zwitterionic Polymer Ionic Liquid for Highly Selective Isolation of Glycoprotein Zhi-Yong Guo,† Xin Hai,† Yi-Ting Wang,† Yang Shu,‡ Xu-Wei Chen,*,† and Jian-Hua Wang*,† †

Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Box 332, Shenyang 110819, China ‡ Institute of Biotechnology, College of Life and Health Sciences, Northeastern University, Box H006, Shenyang 110169, China S Supporting Information *

ABSTRACT: A novel zwitterionic polymer ionic liquid functionalized magnetic nanospheres, shortly as Fe3O4@PCL-PILs, is synthesized by grafting ionic liquid VimCOOHBr onto polymer ε-caprolactone (PCL) modified magnetic nanospheres via esterification and surface-initiated free radical polymerization. This established synthesis strategy offers the obtained magnetic nanospheres with well-defined core−corona structure, compact grafting layer, favorable zwitterionic and negativecharged surface, and high magnetic susceptibility. The as-prepared Fe3O4@PCL-PILs nanospheres exhibit typical “zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC)” behaviors toward protein binding, and selectively adsorption of glycoprotein is achieved. The adsorption capacity of the magnetic nanospheres toward Immunoglobulin G is high up to 1136.4 mg g−1, and the captured Immunoglobulin G could be efficiently recovered by using 0.5% NH3 H2O (v/v) as stripping reagent, providing a recovery of 80.5%. Fe3O4@PCL-PILs nanospheres are then employed as sorbent for the selective isolation of Immunoglobulin G from human whole blood, obtaining high-purity Immunoglobulin G as demonstrated by polyacrylamide gel electrophoresis assays.

■

modules.15 Herein, the new methods to fabricate functional nanomaterials with multidimensional interaction model and high grafting efficiency have been arousing widespread concerns.10,16−18 Recently, hydrophilic interaction liquid chromatography (HILIC) has become a popular method for proteins and peptides enrichment due to the advantages of high selectivity and great reproducibility. Various HILIC materials including zwitterionic-based materials, amide-based materials, and saccharide-based HILIC material19−21 have been reported and demonstrated their efficiencies in protein adsorption. Among the HILIC materials, zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, that is, the derivatized sulfobetaine zwitterionic materials and amino acids zwitterionic materials,22,26−28 show the fascinating performance in the analysis of protein glycosylation. The ZIC-HILIC materials are uniquely characterized by the positive and negative charge and hydrophilic moieties on its surface.22 The ratio of positive and negative charge are balanced into a proximity stoichiometric point,23 which result in weaken electrostatic interactions and

INTRODUCTION The adsorption, isolation, and detection of trace protein and low-abundant biomarkers in the early diagnosis of disease is of great significance.1−3 In the past decades, numerous methodologies based on the antibody−antigen interaction, hydrophilic and hydrophobic interaction, metal chelating interaction, electrostatic interaction, and multimode synergistic effect for protein adsorption and isolation have been reported.4−9 In these isolating strategies, the main interacting sites for protein binding are confined on the surface of solid substrates, particularly for monolayer modification adsorbent. Meanwhile, the efficiency for the isolation of protein or peptide from real samples is heavily affected by the signal interaction pattern nanomaterials.10 And that the application scopes are limited to a narrow range as the various essential properties of different nonomaterials. Accordingly, the combination of different function modules with various interactive models is highly demanded to match the requirement of rapid developed proteomics.11−14 Therefore, complicated modifications have been adopted to endow the final nanomaterials with different functional moieties, in which hybrid synthetic techniques are prerequisited for diverse grafting. However, the intricate synthetic methods are always accompanied by tedious experimental procedure, and low coverage of functional © XXXX American Chemical Society

Received: August 26, 2017 Revised: November 16, 2017 Published: November 27, 2017 A

DOI: 10.1021/acs.biomac.7b01231 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Reagent Co. Ltd. (Shanghai, China). ε-Caprolactone is bought from Aladdin Chemical Reagent Co. Ltd. (Shanghai, China). Dimethyl aminopyridine (DMAP) is purchased from Alfa Aesar Chemical Reagent Co. Ltd. (Shanghai, China). All other reagents are at least analytical-reagent grade and without further purification. Synthesis of Magnetic Fe3O4 Nanoparticles and Fe3O4@PCL Nanoparticles. The magnetic Fe3O4 nanoparticles (Fe3O4 NPs) are synthesized according to previous work.36 Briefly, FeCl3·6H2O (5 mM), C2H5ONa (47 mM) and trisodium citrate (1.3 mM) are dissolved in 70 mL of ethylene glycol. The mixture is stirred vigorously at 80 °C to form a homogeneous claybank solution, and then transferred into 100 mL stainless-steel autoclave for heating at 200 °C for 16 h. The product is cooled down to room temperature, collected under a magnet and washed for several times with ethanol, and then dried at 60 °C in vacuum. The Fe3O4 NPs are then modified with εcaprolactone to produce adequately hydroxyl and hydrophobic chain on the surface according to a reported method.37 Typically, 100 mg Fe3O4 NPs are dispersed into 60 mL of DMF and ultrasonic dispersed to achieve a uniformed suspension. After that, ε-caprolactone (0.077 mM, 3 mL) and stannous octoate (0.006 mM, 1 mL) are added into the mixture and heated in an oil bath at 140 °C for 6 h under reflux. Finally, the product is collected by a magnet, washed with ethanol and dried in vacuum at 60 °C. Preparation of Fe3O4@PCL-VimCOOHBr Nanospheres. Carboxylation ionic liquid (VimCOOHBr) is fabricated according to the reported literature.38 Typically, 1-vinylimidazole (11 mM, 1.2 mL) is added into a mixture containing bromoacetic acid (12 mM, 1.69 g) and acetonitrile (20 mL) drop-wisely under stirring. The reaction mixture are heated to 70 °C and maintained for 3 h with reflux. The obtained white powder is filtered and washed with acetonitrile for several times, and dried at 60 °C vacuum. Then modification of Fe3O4@PCL nanoparticles is achieved through esterification reaction between carboxyl and hydroxyl groups in DMF. Specifically, Fe3O4@PCL nanoparticles (100 mg) are ultrasonic dispersed in a mixture containing DMF (50 mL), DCC (0.8 mM), DMAP (0.12 mM), and VimCOOHBr (0.75 mM). Then the mixture are stirred and reacted for 24 h at room temperature. The obtained Fe3O4@PCL-VimCOOHBr nanospheres are collected by magnet, washed with DMF and ethanol, and stored for further usage. Fabrication of Fe3O4@PCL-PILs Core−Corona Nanospheres. The resultant Fe3O4@PCL-VimCOOHBr nanospheres are used for the surface-initiated free radical polymerization (SI-FRP) to fabricate the Fe3O4@PCL-PILs nanospheres. Fe3O4@PCL-VimCOOHBr nanospheres (100 mg) are dispersed in 60 mL of DMF, then VimCOOHBr (1 mM dissolved in 5 mL DMF) and AIBN (0.05 mM) are added into the suspension under stirring. After bubbling with nitrogen gas for 5 min, the reaction mixture are heated to 70 °C and polymerized at this temperature for 3 h under nitrogen protection. The obtained product is separated by a magnet, washed with ethanol and water in turn, and dried in a vacuum oven at 60 °C. Characterization of the Fe3O4@PCL-PILs Nanospheres. Transmission electron microscopy (TEM) images are taken on a G 2 (Technai, Italia) transmission electron microscope at an accelerating voltage of 200 kV. The samples are dispersed at a suitable concentration and placed onto a carbon-coated copper grid. X-ray diffraction (XRD) patterns are collected on an X’Pert Pro (PANalytical BV, Holland) diffraction meter with Cu Kα, λ = 0.154 nm. Fourier transform infrared spectra (FT-IR) are recorded on a Nicolet 6700 spectrometer (Thermo Electron, U.S.A.) with the scanned range of 4000 to 400 cm−1 and resolution of 2.0 cm−1. Thermogravimetric analysis is conducted on a TGA 290C analyzer (TGA, Netzsch Company, Germany). All experiments temperature is increased from 20 to 1000 °C at a rate of 10 °C min−1 under nitrogen atmospheres. Magnetic characterization is tested on a vibrating sample magnetometer (VSM, Lake Shore, U.S.A.) on a Model 6000 physical property measurement system at 300 K. The pH is adjusted by a pH Meter (Beijing Sartorius Instruments Co., Ltd., China). Hydrodynamic diameter (Dh) and Zeta potential measurements are carried out by dynamic light scattering (DLS) with a Nano ZS90 (Malvern, U.K.).

tremendously strengthen hydrophilic interaction between stationary phase and glycoprotein.24,25 The abundant zwitterionic and hydrophilic groups could reinforce the concentrating of glycoprotein in water layer, making the ZIC-HILIC material a good candidate for glycoprotein enrichment. Ionic liquids (ILs) are a series of materials with unique physicochemical properties, that is, incombustibility, low volatility, high thermal stability, ease of synthesis, and modification. These outstanding characteristics of ILs render them multipurpose in various fields, such as solvent/solid phase extraction, electro-analytical chemistry, chromatography stationary phases, and sensors.29−31 According to the flexible selection of various anions and cations, ionic liquids are provided with diversely physicochemical properties to satisfy specific requirements.32,33 Ionic liquids with imidazole cationic structure endow them with outstanding retention mechanism of HILIC, and have attracting application in the separating water-soluble and polar analyte.34 Recently, zwitterionic ionic liquids are synthesized through the substitution reaction between imidazole cationic and sulfonate and amino acid anion group. The obtained zwitterionic ionic liquids usually show desirable analysis performance in a wide range of polar solutes.35 Hence, we have introduced 1-vinylimidazole as imidazole cationic and bromoacetic acid as anion group to prepare the zwitterionic ionic liquid and applied in the isolation of glycoprotein. In the present study, a novel ZIC-HILIC magnetic nanospheres (Fe3O4@PCL-PILs) are successfully synthesized through the growth of branched polymer ε-caprolactone (PCL) polymer brushes on the surface of Fe3O4 nanoparticles and the subsequent coating with zwitterionically and hydrophilicity polymer ionic liquid (PIL). The hydroxyl groups on the end of PCL chain are converted to double bond groups by esterification with VimCOOHBr. The PCL brushes not only are able to provide enhanced grafting site for VimCOOHBr, but also lead to three-dimensional scaffolds for the immobilization of zwitterionical and hydrophilic PIL. The as-prepared magnetic nanospheres are characterized with defined core− corona structure and compact PIL layer. The zwitterionic properties offer the Fe3O4@ PCL-PILs nanospheres favorable performance on the binding of glycoprotein, and selective separation of Immunoglobulin G (Ig-G) from human whole blood is achieved by using the magnetic nanospheres as sorbent, suggesting the-great potential of this novel ZIC-HILIC magnetic nanospheres in the sample pretreatment of biological samples.

■

EXPERIMENTAL SECTION

Chemicals and Reagents. Immunoglobulin G (Ig-G, 14 506, >95%, Mr 150.0 kDa), hemoglobin (Hb, H2625, >95%, Mr 66.0 kDa), bovine serum albumin (BSA, A 3311, >98%, Mr 66.7 kDa), ovalbumin (Ova, A5503, >98%, Mr 44.3 kDa), cytochrome c (Cyt-c, 30398, >95%, Mr 12.4 kDa), transferrin (Trf, T3309, >98%, Mr 78 kDa), myohemoglobin (Mb, M8007, >95%, Mr 16.7 kDa), and ammonium bicarbonate (ABC, >99%) are obtained from Sigma-Aldrich (St. Louis, U.S.A.). Iron(III) chloride hexahydrate (FeCl3·6H2O), sodium chloride (NaCl), sodium acetate (CH3COONa), trisodium citrate (Na3(cit)), bromoacetic acid, stannous octoate, dicyclohexylcarbodiimide (DCC), ethylene glycol (EG), anhydrous ethanol (≥99.7%), acetonitrile (ACN, ≥99.8%), N,N-dimethylformamide (DMF), ammonium hydroxide (NH3·H2O, 25−28%), cetyltrimethylammonium bromide (CTAB), and sodium dodecyl sulfate (SDS) are purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). 1-Vinylimidazole is purchased from Cheng Jie Chemical B

DOI: 10.1021/acs.biomac.7b01231 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules Isolation and Enrichment of Glycoprotein with Fe3O4@PCLPILs Core−Corona Nanospheres. The obtained Fe3O4@PCL-PILs core−corona nanospheres are prewashed with water and then suspended in deionized water (10 mg mL−1). A total of 1 mL of protein sample (prepared in pH 9 ammonium bicarbonate buffer (ABC buffer)) and 100 μL of Fe3O4@PCL-PILs suspension are mixed together and the mixture is shook on an oscillator for 10 min. After that, Fe3O4@PCL-PILs nanospheres with captured proteins are separated by a magnet and prewashed with pH 9 ammonium bicarbonate buffer. The residual protein content in the solution after capture is quantified by monitoring the characteristic absorption of protein, and the adsorption efficiency is defined as the ratio of the amount of captured protein and that in original protein sample. Subsequently, 1000 μL of 0.5% NH3·H2O is mixed with Fe3O4@PCLPILs nanospheres, and the suspension is shook on an oscillator for 30 min at room temperature to recover the adsorbed proteins. The recovery is thus evaluated by measuring the protein concentration in the recovery solutions. The protein solutions (including the protein sample, sample after adsorption and recovery solutions) are all collected for SDS-PAGE analysis. The dynamic adsorption of proteins onto Fe3O4@PCL-PILs nanospheres at room temperature are performed within the protein concentration range of 10−2500 mg L−1. The adsorption capacity is thus derived from the adsorption curve between protein concentrations and the adsorbed amount of protein.

hydrophilic PILs layer is able to provide more binding sites for protein adsorption. The typical TEM images of Fe3O4 NPs, Fe3O4@PCL, and Fe3O4@PCL-PILs core−corona nanospheres are illustrated in Figure 1a−c. The Fe3O4 NPs present uniformly as spherical and well-dispensed nanoparticles with size of about 175 nm. After coating with PCL, the size of the Fe3O4@PCL nanospheres is estimated to be about 275 nm. The esterification and surface-initiated free radical polymerization with VimCOOHBr result in a core−corona structure of Fe3O4@PCL-PILs nanospheres. The embedded image in Figure 1c evidently reveals a clear Fe3O4 core and a PILs corona of 200 nm in thickness. The enormous increasing thickness of PILs shell indicates that larger proportion of PILs chains have been decorated onto the surface of nanoparticles and the ZIC-HILIC material is successfully achieved. The size change induced by grafting and polymerizing and size distribution in each step also can be monitored by dynamic light scattering. The hydrodynamic diameters of Fe3O4 NPs, Fe3O4@PCL, and Fe3O4@PCL-PILs core−corona nanospheres increase with the modification precess (Figure 1d−f) and agrees well with the results of TEM. Moreover, the polydispersity indexes of Fe3O4 NPs, Fe3O4@PCL, and Fe3O4@PCL-PILs core−corona nanospheres are derived to be 0.152, 0.218, and 0.232, respectively, indicating that the obtained nanospheres all are well-dispersed. The efficient functionalization of PCL and VimCOOHBr to the surface of Fe3O4 nanoparticles is confirmed by FT-IR analysis (Figure 2a). The strong peak at 592 cm−1 appeared in all FT-IR spectra belong to the characteristic stretching vibration of FeO of Fe3O4 NPs. For the citrate-stabilized Fe 3 O 4 NPs, the characteristic peaks of symmetric or antisymmetric COO− and O−H are stretched at 1420, 1605, and 3430 cm−1, respectively. In the spectrum of Fe3O4@PCL nanospheres, the presence of characteristic peaks of PCL at 2851 cm−1, 2928 cm−1 (C−H stretching), and 1720 cm−1 (C O stretching) and the increase of O−H peaks intensity indicates the successful grafting.39 When esterification reaction take place and VimCOOHBr is introduced at the end of the PCL chain, the adsorption peaks of imidazolium cation appear at 1574, 1450, and 1160 cm−1, and the adsorption peak of C C appears at 1627 cm−1. After SI-FRP reaction with VimCOOHBr, the peak at 1632 cm−1 and broad absorption band around 3400−2800 cm−1 are confirmed to the characteristic peaks of COOH in Fe3O4@PCL-PILs core−corona nanospheres. XRD measurements are employed to determine the composition of the obtained magnetic nanospheres. All the diffraction peaks in Figure 2b (i) are indexed and indentified as the typical cubic structure of Fe3O4 (JCPDS 89-0688). For the Fe3O4@PCL and Fe3O4@PCL-PILs nanospheres, the diffraction peaks consisted of 30.2°, 35.6°, 43.1°, 53.5°, 56.9°, and 62.6° correspondes to (220), (311), (400), (422), (511), and (440) planes of Fe3O4, suggesting that the crystallinity of Fe3O4 nanoparticles has remained very well after grafting and polymerizing. Energy dispersive X-ray (EDX) and thermogravimetric analysis (TGA) experiments are carried out to verify the elementary composition of the magnetic nanospheres. As shown in Table 1, the Fe, O, and C are the basic elements of Fe3O4 NPs. After functionalization with PCL layer, the main elements remain unchanged, while the individual contents are slightly changed due to the coating of PCL. For the Fe3O4@

■

RESULTS AND DISCUSSION Preparation of Magnetic Fe3O4@PCL-PILs Core− Corona Nanospheres. The synthesis routes of the magnetic ZIC-HILIC nanomaterials, including the modification of magnetic Fe3O4 core, the cross-linking of intermediate VimCOOHBr layer and the formation of PILs shell are schematically illustrated in Scheme 1. The magnetic core Fe3O4 Scheme 1. Schematic Illustration of Synthetic Procedure for the Fe3O4@PCL-PILs Core−Corona Nanospheres

NPs is first synthesized through a modified solvothermal reaction, then a tight PCL layer is coated onto the magnetic core via the ring-opening polymerization. The introduction of PCL chain offers the surface of magnetic Fe3O4 with abundant hydroxyl groups. Finally, the zwitterionic and hydrophilic PILs are assembled onto the Fe3O4@PCL surface by esterification and surface-initiated free radical polymerization to generate Fe3O4@PCL-PILs core−corona nanospheres. The adopted preparation route offers the obtained nanospheres not only with a compact grafted layer, but also uniformed size distribution. Such homogeneous shell of zwitterionic and C

DOI: 10.1021/acs.biomac.7b01231 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 1. (a−c) TEM images of Fe3O4 NPs, Fe3O4@PCL, and Fe3O4@PCL-PILs core−corona nanospheres, respectively. (d−f) Hydrodynamic diameter of Fe3O4 NPs, Fe3O4@PCL, and Fe3O4@PCL-PILs core−corona nanospheres, respectively.

Adsorption Behaviors of Protein on Fe3O4@PCL-PILs Core−Corona Nanospheres. To investigate the performance of the ZIC-HILIC magnetic nanospheres in the adsorption of different protein species, the adsorption behaviors of Ig-G, Trf, and Ova as glycoprotein model and Hb, BSA, Cyt-c, and Mb as the nonglycoprotein model onto the Fe3O4@PCL-PILs core− corona nanospheres are investigated. It can be seen that better adsorption of glycoproteins than nonglycoproteins under same adsorption condition are observed. The difference between glycoprotein and nonglycoprotein can be well explained by the different interactions between protein species and ZIC-HILIC magnetic nanospheres. Glycoprotein is a kind of proteins containing glycan group inside their structure. The glycan group is usually consisted of hydrophilic mannose residues, glucosamine residues, biantennary glycan structures and so on.40 Therefore, there is strong hydrophilic interaction existing between hydrophilic glycans and the hydrophilic surface of ZIC-HILIC nanospheres. Consequently, the Fe3O4@PCL-PILs core−corona nanospheres as a novel ZIC-HILIC material could afford great petential for glycoprotein isolation. It can be seen that the highest adsorption efficiencies, that is, 82.3% for Ova, 86% for Ig-G, and 34.2% for Trf are achieved at different pH (Figure 3a). As typical glycoprotein, Ova are consisted with 4−6 mannose residues and 2−4-glucosamine residues and Ig-G are included 30 different biantennary glycan structures.40 Trf contains two glycosylation sites at Asn413 and Asn611 with two biantennary N-acetyllactosamine residues and four terminal sialic acid residues. The glycan groups are usually hided in the hydrophobic cavity of protein and exposed at a pH environment condition near to the isoelectric points of protein. Though, the best adsorption efficiency of nonglycoprotein Hb, BSA, Cyt-c, and Mb are also achieved at the pH conditions near to their pI, while the adsorption efficiencies are much lower than that of glycoproteins under their optimal condition. Above all, satisfactory adsorption of Ig-G is obtained at pH 9, while actually no adsorption of BSA, Mb, Cyt-c, and Hb takes place at the same conditions. These above results suggest that the as-prepared ZIC-HILIC nanospheres can be used for efficient adsorbent for the

PCL-PILs core−corona nanospheres, the newly appearance of element N indicates the successful grafting and polymerizing of PILs onto the surface of Fe3O4@PCL nanospheres. The TGA curves show a weight loss about 6.3 wt % for Fe3O4 NPs (Figure 2c(i)), attributed to the decomposition of citrate. After grafting with PCL and PILs, the weight loss of Fe3O4@PCL and Fe3O4@PCL-PILs core−corona nanospheres is about 13.3% and 38.9 wt %, respectively, demonstrating the weight loss of PILs layer is about 25.6% and presence of a high density PILs shell on Fe3O4@PCL nanospheres. The magnetic properties of the magnetic nanospheres are measured by using a vibrating sample magnetometer (VSM). As shown in Figure 2d, the magnetic hysteresis loops (Hc < 30 Oe) are very weakly which demonstrating that these magnetic materials are superparamagnetic under ambient condition. The saturation magnetization (Ms) value of Fe3O4 NPs is 62.5 emu g−1. After coating with PCL layer, the Ms value is reduced to 52.4 emu g−1. Upon the esterification and SI-FRP reaction grafting with PILs shell, the Ms value bring about an abrupt changing to 36.8 emu g−1. The magnetic responsiveness of the Fe3O4@PCL-PILs nanospheres is sufficient to promote the separation of magnetic nanospheres from solution using a magnet. According to the saturation magnetization of the nanospheres, the content of PILs is achieved to be about 29.7% in the Fe3O4@PCL-PILs core−corona nanospheres, which fits well with that obtained from the results of TGA. Figure 2e illustrates the zeta potential of these nanopheres. The surface of Fe3O4 NPs and Fe3O4@PCL nanospheres both are negatively charged. After esterification and SI-FRP polymerization with ionic liquid, the zeta potential of the final nanospheres converts to 5.5 mV and −33.8 mV, respectively. The changes of zeta potential well demonstrate the successful decoration of PILs shell onto the Fe3O4@PCL nanospheres. The surface-charge properties of the Fe3O4@ PCL-PILs nanospheres in aqueous solution are shown in Figure 2f. Owing to the carboxylic acid group on the surface of nanospheres, which are identical with the typical ZIC-HILIC materials.39 Fe3O4@PCL-PILs nanospheres is negatively charged in the tested pH range from 3 to 10. D

DOI: 10.1021/acs.biomac.7b01231 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 2. FT-IR spectra (a), XRD patterns (b), TGA curves (c), VSM curves (d), and Zeta potentials of Fe3O4 NPs (i), Fe3O4@PCL nanospheres (ii), Fe3O4@PCL-VimCOOHBr nanospheres (iii), and Fe3O4@PCL-PILs core−corona nanospheres (iv) (e). Zeta potentials of the Fe3O4@PCLPILs nanospheres at pH 3−10 (f).

adsorption efficiency. These phenomena might be well explained by hydrophilic interaction between the ZIC-HILIC magnetic nanospheres and the protein molecules. The increase of salinity will cause the breakage of water clusters around ZICHILIC magnetic nanospheres and protein molecules,42 and the outspread of hydrophilic PIL chains, which facilitates the binding of Ig-G molecules onto ZIC-HILIC magnetic nanospheres and eventually contributes to the increasing of adsorption efficiency. The protein adsorption capacity by the Fe3O4@PCL-PILs core−corona nanospheres is investigated at room temperature within a series of protein solutions from 10 to 2500 mg L−1. The adsorption curve is obtained by plotting protein concentrations versus the adsorbed amount of protein (Figure S1). It is obvious that the adsorption behavior of protein onto the Fe3O4@PCL-PILs core−corona nanospheres fits well with Langmuir model as showed in the following equation, where C* is the concentration of protein, Q* represents the amount of protein captured with the Fe3O4@PCL-PILs core−corona nanospheres, Qm is the maximum adsorption capacity, and Kd is the dissociation constant. By fitting the experimental data to

Table 1. Element Counts of Fe3O4 NPs, Fe3O4@PCL, and Fe3O4@PCL-PILs Nanospheres Derived from EDX Spectra element counts (%) materials

Fe

O

C

N

Fe3O4 Fe3O4@PCL Fe3O4@PCL-PILs

56.852 53.823 41.282

39.910 41.352 39.283

3.238 4.825 15.355

3.620

selectively isolation of glycanprotein. To further confirm the adsorption mechanism of Ig-G is dominant by hydrophilic interaction, the adsorption behaviors of Ig-G on the nanospheres are detailedly studied. Figure 3b illustrates the influence of the ionic strength on the adsorption of Ig-G with NaCl concentration ranging from 0 to 500 mM. The adsorption efficiency of Ig-G first increase with NaCl concentration in the range of 0−400 mM, then level off when the NaCl concentration keeps further increasing. However, the electrostatic are extremely inhibited while the protein adsorbed onto the surface of the ZIC-HILIC material.41 So the electrostatic interaction is not the decisively factor for the increasing of the E

DOI: 10.1021/acs.biomac.7b01231 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 3. (a) Adsorption efficiency of glycoprotein Ig-G, Ova, and Trf, and nonglycoprotein Hb, BSA, Cyt-c, and Mb onto the surface of Fe3O4@ PCL-PILs nanospheres. (b) Ionic strength-dependent adsorption behaviors of Ig-G onto the Fe3O4@PCL-PILs nanospheres. The protein solution, 1.0 mL (the concentration of each protein is 100 mg L−1); pH 5, 7, and 9; adsorption time, 10 min; the mass of nanospheres, 1.0 mg. All the results are from three repeated determinations.

Recovery of the Retained Ig-G from Fe3O4@PCL-PILs Core−Corona Nanospheres. For ensuring biological investigations and practical application, it is essential to recovery the adsorbed protein from magnetic nanospheres into an aqueous medium. Therefore, the recovery of retained protein from Fe3O4@PCL-PILs core−corona nanospheres surface is investigated with a series of potential stripping reagents (Figure 4a), including CTAB, imidazole, NH3 H2O, BR buffers, citrate, and tris-HCl. The results show that NH3 H2O offers favorable recovery for Ig-G. For further investigations, a series of NH3 H2O solution (concentration range from 0.1% to 1.0% (v/v)) are tested for the recovery of retained proteins, 0.5% (v/v) NH3 H2O, giving rise to recovery efficiency to 80.5% for Ig-G. This might be ascribed to the high pH environment caused a seriously break down of the hydrophilic interaction.47 The reusability of the ZIC-HILIC magnetic nanospheres is investigated by performing continuous adsorption−desorption process. The results (Figure S2) illustrate that virtually no obvious change on the adsorption efficiency of Ig-G is observed even after 10 continuous adsorption/desorption process, indicating the favorable reusability of Fe3O4@PCL-PILs core−corona nanospheres. Isolation of Ig-G from Human Whole Blood with Fe3O4@PCL-PILs Core−Corona Nanospheres. The practical application of Fe3O4@PCL-PILs core−corona nano-

this equation, the maximum theoretical adsorption capacity for Ig-G is derived to be 1136.4 mg g−1. Obviously, the adsorption capability of Fe3O4@PCL-PILs nanospheres toward Ig-G is superior to other nanomaterials (Table 2).43−46 The superiority Table 2. Comparison of Adsorption Capacities for Immunoglobulin G with Various Adsorbents adsorbents

adsorption capacity (mg g−1)

ref

Mo8O26@MIL-101(Cr) octapeptide affinity resins PHEMA/PGMA-IDA-Cu2+ His-MWNTs Fe3O4@PCL-PILs nanospheres

596.6 176.4 257.0 267.0 1136.4

43 44 45 46 this work

of Fe3O4@PCL-PILs nanospheres on Ig-G adsorption is ascribed to the compact polymer ionic liquid layer. This abundant zwitterionic molecules of compact polymer ionic liquid layer not only offers the obtained nanospheres enhanced hydrophilicity but also provides it improved binding sites for protein adsorption when compare other materials.

Q* =

Q m × C* Kd + C *

Figure 4. (a) Recovery of the retained Ig-G from the Fe3O4@PCL-PILs nanospheres with different stripping reagents. The concentration of CTAB and NH3 H2O is 0.5 mg mL−1. The concentration of citrate, imidazole, BR, and tris-HCL is 0.1 mol L−1. (b) Elution efficiency of the adsorbed Ig-G with different concentration of NH3 H2O (the results from triplicate determinations). Protein solution, 1.0 mL, 100 mg L−1, pH 9; adsorption time, 10 min; magnetic nanospheres, 1.0 mg; elution time, 30 min. F

DOI: 10.1021/acs.biomac.7b01231 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Fe3O4@PCL-PILs nanospheres as sorbent well suggests the great practicability of Fe3O4@PCL-PILs nanospheres in the pretreatment of biological samples. It can be expected that the new course will increase the coverage of functional modules and improve the enrichment efficiency. And the as-obtained Fe3O4@PCL-PILs core−corona nanospheres will hold great potential in glycoproteomics research and hydrophilic interaction chromatography.

spheres is performed by the selective separation of Ig-G from human whole blood. The human whole blood sample anticoagulated with sodium citrate is donated by the Hospital of Northeastern University. The blood sample is diluted for 100-fold with 50 mmol L−1 ABC buffer (pH 9) and mixed with Fe3O4@PCL-PILs nanospheres (1 mg) for undergoing the adsorption process, as described in the Experimental Section. Afterward, the adsorbed Ig-G is recovered from the ZIC-HILIC material with a 0.5% NH3 H2O solution. For SDS-PAGE assay, the recovered sample solution and standard sample is mixed with loading buffer and boiling for 10 min. Then the electrophoresis experiments are demonstrated on 5% polyacrylamide stacking gel and 12% polyacrylamide separation gel at 90 and 180 V, respectively. The separation gel are stained with 0.1% (w/v) coomassie brilliant blue G-250, and destained with a mixture solution containing 7.5% (v/v) acetic acid and 5% (v/v) methanol. The SDS-PAGE assay results are showed in Figure 5. The protein bands in lane 2 are consisted mainly within HSA (66.4

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b01231. The adsorption isotherm of Ig-G, BSA, Hb, cyt-c, Mb, Trf and Ova on Fe3O4@PCL-PILs nanospheres. Protein solution, 10−2500 mg L−1, pH 9; adsorption time, 10 min; the mass of nanospheres, 1.0 mg. The reusability of Fe3O4@PCL-PIL nanospheres for the circulation of adsorption and desorption of Ig-G. Protein solution, 1.0 mL, 100 mg L−1, pH 9; adsorption time, 10 min; magnetic nanospheres, 1.0 mg (PDF).

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Tel.: +86 24 83688944; Fax: +86 24 83676698. ORCID

Yang Shu: 0000-0002-1596-5943 Xu-Wei Chen: 0000-0001-7189-5022 Jian-Hua Wang: 0000-0003-2175-3610

Figure 5. SDS-PAGE assay results: lane 1, molecular weight standard (markers are in kDa); lane 2, 100-fold diluted human whole blood without pretreatment; lane 3, 100-fold diluted human whole blood after adsorption by the Fe3O4@PCL-PILs nanospheres; lane 4, Ig-G recovered from the Fe3O4@PCL-PILs nanospheres; lane 5, Ig-G standard solution (150 mg L−1).

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors appreciate financial support from the Natural Science Foundation of China (21475017, 21275027, and 21235001) and Fundamental Research Funds for the Central Universities (N150502001).

kDa), Ig-G (50.0 kDa), carbonic anhydrase (29.0 kDa), Hb (14.3 kDa), and Cyt-c (6.5 kDa). After adsorbed with Fe3O4@ PCL-PILs nanospheres and recovery with 0.5 mL of 0.5% NH3 H2O solution, the bands of Ig-G (50.0 kDa) is still observed, while the band of HSA, carbonic anhydrase, Hb, and Cyt-c disappear (lane 4). The left band in lane 4 is identified at the same position as that of 150 mg L−1 Ig-G standard solution (lane 5). The higher performance adsorption of Ig-G from human whole blood with the Fe3O4@PCL-PILs nanospheres illustrated the effective isolation material ZIC-HILIC magnetic nanospheres could exclude obstacles in the presence of other protein species and apply in the complex sample matrixes.

■

REFERENCES

(1) Issaq, H. J.; Xiao, Z.; Veenstra, T. D. Serum and Plasma Proteomics. Chem. Rev. 2007, 107, 3601−3620. (2) Cheng, G.; Zhou, M. D.; Zheng, S. Y. Facile Synthesis of Magnetic Mesoporous Hollow Carbon Microspheres for Rapid Capture of Low-Concentration Peptides. ACS Appl. Mater. Interfaces 2014, 6, 12719−12728. (3) Liu, Z.; Fan, S. H.; Liu, H. P.; Yu, J.; Qiao, R.; Zhou, M.; Yang, Y. T.; Zhou, J.; Xie, P. Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion. PLoS One 2016, 11, e0166306. (4) Casalini, S.; Dumitru, A. C.; Leonardi, F.; Bortolotti, C. A.; Herruzo, E. T.; Campana, A.; Oliveira, R. F.; Cramer, T.; Garcia, R.; Biscarini, F. Multiscale Sensing of Antibody Antigen Interactions by Organic Transistors and Single-Molecule Force Spectroscopy. ACS Nano 2015, 9, 5051−5062. (5) Wei, Q.; Becherer, T.; Uberti, S. A.; Dzubiella, J.; Wischke, C.; Neffe, A. T.; Lendlein, A.; Ballauff, M.; Haag, R. Protein Interactions with Polymer Coatings and Biomaterials. Angew. Chem., Int. Ed. 2014, 53, 8004−8031. (6) Liu, J. W.; Zhang, Y.; Chen, X. W.; Wang, J. H. Graphene OxideRare Earth Metal-Organic Framework Composites for the Selective

■

CONCLUSIONS In summary, a novel magnetic ZIC-HILIC nanospheres containing compact corona-like PILs shell are prepared via esterification and surface-initiated free radical polymerization. The compact polymer ionic liquid layer provides the obtained nanospheres enhanced hydrophilicity and abundant zwitterionic molecules on the magnetic nanospheres. The as-prepared Fe3O4@PCL-PILs core−corona nanospheres have been demonstrated to be able to provide favorable adsorption specificity and high binding capacity toward glycoprotein. The successful selective separation of Ig-G from human whole blood with the G

DOI: 10.1021/acs.biomac.7b01231 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules Isolation of Hemoglobin. ACS Appl. Mater. Interfaces 2014, 6, 10196− 10204. (7) Hwang, L.; Guner, S. A.; Gregorich, Z. R.; Cai, W. X.; Valeja, S. G.; Jin, S.; Ge, Y. Specific Enrichment of Phosphoproteins Using Functionalized Multivalent Nanoparticles. J. Am. Chem. Soc. 2015, 137, 2432−2435. (8) Moerz, S. T.; Huber, P. Protein Adsorption into Mesopores: A Combination of Electrostatic Interaction, Counterion Release and Vander Waals Forces. Langmuir 2014, 30, 2729−2737. (9) Zhang, Y.; Xing, L. G.; Chen, X. W.; Wang, J. H. Nano Copper Oxide-Incorporated Mesoporous Carbon Composite as Multimode Adsorbent for Selective Isolation of Hemoglobin. ACS Appl. Mater. Interfaces 2015, 7, 5116−5123. (10) Zhang, Y. T.; Yu, M.; Zhang, Z. H.; Guo, J.; Wang, C. C. Magnetic Nano-Sponges for High-Capacity Protein Enrichment and Immobilization. Small 2016, 12, 4815−4820. (11) Li, Y.; Zhang, X. M.; Deng, C. H. Functionalized Magnetic Nanoparticles for Sample Preparation in Proteomics and Peptidomics Analysis. Chem. Soc. Rev. 2013, 42, 8517−8539. (12) Chen, Y. J.; Xiong, Z. C.; Peng, L.; Gan, Y. Y.; Zhao, Y. M.; Shen, J.; Qian, J. H.; Zhang, L. Y.; Zhang, W. B. Facile Preparation of Core-Shell Magnetic Metal-Organic Framework Nanoparticles for the Selective Capture of Phosphopeptides. ACS Appl. Mater. Interfaces 2015, 7, 16338−16347. (13) Zhang, Y. T.; Ma, W. F.; Li, D.; Yu, M.; Guo, J.; Wang, C. C. Benzoboroxole Functionalized Magnetic Core/Shell Microspheres for Highly Specific Enrichment of Glycoproteins under Physiological Conditions. Small 2014, 10, 1379−1386. (14) Zhang, Y. W.; Zhang, M.; Yang, J. B.; Ding, L.; Zheng, J.; Xu, J. L.; Xiong, S. L. Formation of Fe3O4@SiO2@C/Ni Hybrids with Enhanced Catalytic Activity and Histidine-Rich Protein Separation. Nanoscale 2016, 8, 15978−15988. (15) Ma, W. F.; Zhang, Y.; Li, L. L.; Zhang, Y. T.; Yu, M.; Guo, J.; Lu, H. J.; Wang, C. C. Ti4+ -Immobilized Magnetic Composite Microspheres for Highly Selective Enrichment of Phosphopeptides. Adv. Funct. Mater. 2013, 23, 107−115. (16) Liu, J. X.; Yang, K. G.; Shao, W. Y.; Qu, Y. Y.; Li, S. W.; Wu, Q.; Zhang, L. H.; Zhang, Y. K. Boronic Acid-Functionalized Particles with Flexible Three-Dimensional Polymer Branch for Highly Specific Recognition of Glycoproteins. ACS Appl. Mater. Interfaces 2016, 8, 9552−9556. (17) Shao, M. F.; Ning, F. Y.; Zhao, J. W.; Wei, M.; Evans, D. G.; Duan, X. Preparation of Fe3O4@SiO2@Layered Double Hydroxide Core-Shell Microspheres for Magnetic Separation of Proteins. J. Am. Chem. Soc. 2012, 134, 1071−1077. (18) Xiong, Z. C.; Zhao, L.; Wang, F. J.; Zhu, J.; Qin, H. Q.; Wu, R. A.; Zhang, W. B.; Zou, H. F. Synthesis of Branched PEG Brushes Hybrid Hydrophilic Magnetic Nanoparticles for the Selective Enrichment of N-linked Glycopeptides. Chem. Commun. 2012, 48, 8138− 8140. (19) Tang, F.; Yu, Q. W.; Yuan, B. F.; Feng, Y. Q. Hydrophilic Materials in Sample Pretreatment. TrAC, Trends Anal. Chem. 2017, 86, 172−184. (20) Liu, J. X.; Yang, K. G.; Shao, W. Y.; Li, S. W.; Wu, Q.; Zhang, S.; Qu, Y. Y.; Zhang, L. H.; Zhang, Y. K. Synthesis of Zwitterionic Polymer Particles via Combined Distillation Precipitation Polymerization and Click Chemistry for Highly Efficient Enrichment of Glycopeptide. ACS Appl. Mater. Interfaces 2016, 8, 22018−22024. (21) Lin, H.; Ou, J. J.; Zhang, Z. B.; Dong, J.; Wu, M. H.; Zou, H. F. Facile Preparation of Zwitterionic Organic-Silica Hybrid Monolithic Capillary Column with an Improved “One-Pot” Approach for Hydrophilic-Interaction Liquid Chromatography (HILIC). Anal. Chem. 2012, 84, 2721−2728. (22) Dedvisitsakul, P.; Jacobsen, S.; Svensson, B.; Bunkenborg, J.; Finnie, C. Häg glund, P. Glycopeptide Enrichment Using a Combination of ZIC-HILIC and Cotton Wool for Exploring the Glycoproteome of Wheat Flour Albumins. J. Proteome Res. 2014, 13, 2696−2703.

(23) Cheng, X. D.; Hao, Y. H.; Peng, X. T.; Yuan, B. F.; Shi, Z. G.; Feng, Y. Q. Preparation and Chromatographic Evaluation of Zwitterionic Stationary Phases with Controllable Ratio of Positively and Negatively Charged Groups. Talanta 2015, 141, 8−14. (24) Palma, S. D.; Boersema, P. J.; Heck, A. J. R.; Mohammed, S. Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZICHILIC and ZIC-cHILIC) Provide High Resolution Separation and Increase Sensitivity in Proteome Analysis. Anal. Chem. 2011, 83, 3440−3447. (25) Mysling, S.; Palmisano, G.; Højrup, P.; Andersen, M. T. Utilizing Ion-Pairing Hydrophilic Interaction Chromatography Solid Phase Extraction for Efficient Glycopeptide Enrichment in Glycoproteomics. Anal. Chem. 2010, 82, 5598−5609. (26) Qiao, L. Z.; Shi, X. Z.; Xu, G. W. Recent Advances in Development and Characterization of Stationary Phases for Hydrophilic Interaction Chromatography. TrAC, Trends Anal. Chem. 2016, 81, 23−33. (27) Alswieleh, A. M.; Cheng, N.; Canton, I.; Ustbas, B.; Xue, X.; Ladmiral, V.; Xia, S. J.; Ducker, R. E.; Zubir, O. E.; Cartron, M. L.; Hunter, C. N.; Leggett, G. J.; Armes, S. P. Zwitterionic Poly(amino acid methacrylate) Brushes. J. Am. Chem. Soc. 2014, 136, 9404−9413. (28) Cao, L. W.; Yu, L.; Guo, Z. M.; Shen, A. J.; Guo, Y.; Liang, X. M. N-Glycosylation Site Analysis of Proteins from Saccharomyces cerevisiae by Using Hydrophilic Interaction Liquid ChromatographyBased Enrichment, Parallel Deglycosylation, and Mass Spectrometry. J. Proteome Res. 2014, 13, 1485−1493. (29) Hayes, R.; Warr, G. G.; Atkin, R. Structure and Nanostructure in Ionic Liquids. Chem. Rev. 2015, 115, 6357−6426. (30) Hunt, P. A.; Ashworth, C. R.; Matthews, R. P. Hydrogen Bonding in Ionic Liquids. Chem. Soc. Rev. 2015, 44, 1257−1288. (31) Smiglak, M.; Pringle, J. M.; Lu, X.; Han, L.; Zhang, S.; Gao, H.; MacFarlane, D. R.; Rogers, R. D. Ionic Liquids for Energy, Materials, and Medicine. Chem. Commun. 2014, 50, 9228−9250. (32) Dai, Q.; Ma, J. Q.; Ma, S. Q.; Wang, S. Y.; Li, L. J.; Zhu, X. H.; Qiao, X. Q. Cationic Ionic Liquids Organic Ligands Based MetalOrganic Frameworks for Fabrication of Core-Shell Microspheres for Hydrophilic Interaction Liquid Chromatography. ACS Appl. Mater. Interfaces 2016, 8, 21632−21639. (33) Mao, Q. X.; Wang, W. J.; Hai, X.; Shu, Y.; Chen, X. W.; Wang, J. H. The Regulation of Hydrophilicity and Hydrophobicity of Carbon Dots via a One-pot Approach. J. Mater. Chem. B 2015, 3, 6013−6018. (34) Shu, Y.; Lu, J.; Mao, Q. X.; Song, R. S.; Wang, X. Y.; Chen, X. W.; Wang, J. H. Ionic liquid Mediated Organophilic Carbon Dots for Drug Delivery and Bioimaging. Carbon 2017, 114, 324−333. (35) Qiao, L. Z.; Dou, A. B.; Shi, X. Z.; Li, H.; Shan, Y. H.; Lu, X.; Xu, G. W. Development and Evaluation of New Imidazolium-Based Zwitterionic Stationary Phases for Hydrophilic Interaction Chromatography. J. Chromatogra. A 2013, 1286, 137−145. (36) Guo, Z. Y.; Zhang, Y.; Zhang, D. D.; Shu, Y.; Chen, X. W.; Wang, J. H. Magnetic Nanospheres Encapsulated by Mesoporous Copper Oxide Shell for Selective Isolation of Hemoglobin. ACS Appl. Mater. Interfaces 2016, 8, 29734−29741. (37) Yun, H. M.; Ahn, S. J.; Park, K. R.; Kim, M. J.; Kim, J. J.; Jin, G. Z.; Kim, H. W.; Kim, E. C. Magnetic Nanocomposite Scaffolds Combined with Static Magnetic Field in the Stimulation of Osteoblastic Differentiation and Bone Formation. Biomaterials 2016, 85, 88−98. (38) Liu, J. W.; Wang, M. M.; Zhang, Y.; Han, L.; Chen, X. W.; Wang, J. H. Polymeric Ionic Liquid Modified Reduced Grapheme Oxide as Adsorbent for Highly Selective Isolation of Acidic Protein. RSC Adv. 2014, 4, 61936−61943. (39) Jiang, W.; Fischer, G.; Girmay, Y.; Irgum, K. Zwitterionic Stationary Phase with Covalently Bonded Phosphorylcholine Type Polymer Grafts and Its Applicability to Separation of Peptides in the Hydrophilic Interaction Liquid Chromatography Mode. J. Chromatogr. A 2006, 1127, 82−91. (40) Zhang, Y.; Zhuang, Y. T.; Shen, H. Y.; Chen, X. W.; Wang, J. H. A Super Hydrophilic Silsesquioxane-based Composite for Highly H

DOI: 10.1021/acs.biomac.7b01231 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules Selective Adsorption of Glycoproteins. Microchim. Acta 2017, 184, 1037−1044. (41) Susanto, H.; Ulbricht, M. Photografted Thin Polymer Hydrogel Layers on PES Ultrafiltration Membranes: Characterization, Stability, and Influence on Separation Performance. Langmuir 2007, 23, 7818− 7830. (42) Yang, Q.; Ulbricht, M. Novel Membrane Adsorbers with Grafted Zwitterionic Polymers Synthesized by Surface-Initiated ATRP and Their Salt-Modulated Permeability and Protein Binding Properties. Chem. Mater. 2012, 24, 2943−2951. (43) Chen, Q.; Wang, M. M.; Hu, X.; Chen, X. W.; Wang, J. H. An Octamolybdate-Metal Organic Framework Hybrid for the Efficient Adsorption of Histidine-Rich Proteins. J. Mater. Chem. B 2016, 4, 6812−6819. (44) Zhao, W. W.; Shi, Q. H.; Sun, Y. Dual-Ligand Affinity Systems with Octapeptide Ligands for Affinity Chromatography of HIgG and Monoclonal Antibody. J. Chromatogr. A 2014, 1369, 64−72. (45) Bereli, N.; Şener, G.; Altıntaş, E. B.; Yavuz, H.; Denizli, A. Poly(glycidyl methacrylate) Beads Embedded Cryogels for PseudoSpecific Affinity Depletion of Albumin and Immunoglobulin G. Mater. Sci. Eng., C 2010, 30, 323−329. (46) Du, Z.; Zhang, S. L.; Zhou, C. Y.; Liu, M.; Li, G. K. L-Histidine Functionalized Multi-Walled Carbon Nanotubes form-Line Affinity Separation and Purification of Immunoglobulin G in Serum. Talanta 2012, 99, 40−49. (47) Dong, L. P.; Feng, S.; Li, S. S.; Song, P. P.; Wang, J. D. Preparation of Concanavalin A-Chelating Magnetic Nanoparticles for Selective Enrichment of Glycoproteins. Anal. Chem. 2015, 87, 6849− 6853.

I

DOI: 10.1021/acs.biomac.7b01231 Biomacromolecules XXXX, XXX, XXX−XXX