Facile Synthesis of Mercaptophenylboronic Acid-Functionalized Core

May 3, 2010 - In this work, we reported an alternative strategy to the synthesis of core−shell ..... The bands at 1000−1300 cm−1 corresponding t...
0 downloads 0 Views 4MB Size
Download PDF
J. Phys. Chem. C 2010, 114, 9221–9226

9221

Facile Synthesis of Mercaptophenylboronic Acid-Functionalized Core-Shell Structure Fe3O4@C@Au Magnetic Microspheres for Selective Enrichment of Glycopeptides and Glycoproteins Dawei Qi,† Huaiyuan Zhang,† Jia Tang,† Chunhui Deng,* and Xiangmin Zhang* Department of Chemistry and Institutes of Biomedical Sciences, Shanghai 200433, China ReceiVed: December 2, 2009; ReVised Manuscript ReceiVed: April 8, 2010

In this work, we reported an alternative strategy to the synthesis of core-shell structure Fe3O4@C@Au magnetic microspheres by a self-assembly approach. The as-synthesized Fe3O4@C@Au magnetic microspheres were functionalized with 4-mercaptophenylboronic acid and successfully applied for selective enrichment of glycopeptides and glycoproteins. At first, the Fe3O4@C magnetic microspheres were synthesized by two-step reactions including solvothermal and hydrothermal reactions. Then, Fe3O4@C@Au magnetic microspheres with core-shell structure were obtained by a self-assembly approach. Finally, the Fe3O4@C@Au magnetic microspheres were modified with 4-mercaptophenylboronic acid. The 4-mercaptophenylboronic acid-modified Fe3O4@C@Au magnetic microspheres were successfully applied to selective enrichment of glycoproteins and glycopeptides. 1. Introduction Glycosylation is one of the most important and abundant posttranslational modifications in nature. Glycoproteins play important roles during molecular and cellular recognition in development, growth, and cellular communication and in particular are involved in cancer progression and immune responses. Glycoproteins have been used as therapeutic targets and biomarkers for cancer prognosis, diagnosis, and monitoring.1-6 It is essential to characterize glycoproteins before a full comprehension of the diverse functions of protein can be obtained. The determination of these glycoproteins is still an enduring analytical challenge with current ionization technologies such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). To solve this problem, glycoproteins are selectively cleaved into glycopeptides with unchanged glycosylation sites and other fragments. It is therefore necessary to have an enrichment step of glycopeptides to enhance the detection sensitivity to low-abundant but multiglycosylated proteins prior to MS analysis. Several methods such as lectin-based affinity chromatography, hydrophilic interaction liquid chromatography, and hydrazide chemistry have been widely developed for isolation and identification of N-linked glycopeptides in complex biological samples.7-11 In recent decades, aromatic boronic acid derivatives have been employed in the construction of receptors for molecules such as saccharides. Boronic acids form covalent bonds with cis-diol OHcontaining structures, present in most sugar moieties, to generate five- or six-membered cyclic esters in nonaqueous or basic aqueous media. The boronic acids are readily available, stabile, and versatile, complementing traditional lectins to provide wider sugar binding coverage. Boronic acid affinity chromatography has been employed to isolate glycoproteins, and these methods have been applied in biological samples.12-20 Magnetic nanomaterials have attracted much attention because of the ease of isolation of the magnetic material-target * To whom correspondence should be addressed. Fax: 86-21-65641740. E-mail: [email protected] and [email protected]. † These three authors equally contributed to this work.

conjugate from the sample solution based on their magnetic properties, and have been used in proteomics research and protein separation.21-25 Recently, boronic acid modified magnetic particles were also applied to quickly separate and enrich glycopeptides or glycoproteins for proteomics.12-14 However, modification of boronic acid on magnetic particles requires a tedious multistep chemical reaction. As a result, the amount of boronic acid on magnetic particles is low, which affects the enrichment efficiency for glycoproteins or glycopeptides. As we know, gold nanoparticles show easily tuned physical properties, including unique optical properties, robustness, and high surface areas, making them ideal candidates for developing biomarker platforms. The surface of gold nanoparticles can be tailored by ligand functionalization to selectively bind a target molecule. Many approaches have reported that the surface of gold nanoparticles was functionalized with ligands for specific binding of protein and other target molecules.26-29 More recently, gold nanoparticles were reported to easily immobilize a large amount of 4-mercaptophenylboronic acid due to the robust interaction between the thiol group and gold nanoparticles. Their enrichment efficiency for glycopeptides or glycoproteins has successfully been demonstrated.15,30 Considering the advantages of two different nanomaterials of gold nanoparticles and magnetic particles, the synthesis of Fe3O4@Au for the immobilization of 4-mercaptophenylboronic acid for rapid enrichment of glycoproteins and glycopeptides is very interesting. Many efforts have been made to synthesize the bifunctional materials combining gold and iron oxide. Current synthetic protocols for such bifunctional nanomaterials include, for example, reducing Au3+ onto Fe3O4 nanoparticles surface via iterative hydroxylamine seeding, decomposing Fe(CO)5 on the surface of the Au nanoparticles followed by oxidation in 1-octadecene solvent, and Au3+ reduction onto Fe3O4 nanoparticles deposited on silica cores to form three-layer composite nanoparticles.31-34 Layer-by-layer self-assembly is an effective approach to construction of well-defined nanostructures with functional hybrid nanoshells.35-38

10.1021/jp9114404  2010 American Chemical Society Published on Web 05/03/2010

9222

J. Phys. Chem. C, Vol. 114, No. 20, 2010

Qi et al.

SCHEME 1: The Synthesis Strategy for Boronic Acid-Functionalized Core-Shell Structure Fe3O4@C@Au Magnetic Microspheres

Herein, we represent a facile approach to synthesize core-shell structure Fe3O4@C@Au magnetic microspheres by layer-bylayer self-assembly (Scheme 1), and used for immobilization 4-mercaptophenylboronic acid for selective enrichment of glycoproteins and glycopeptides. First, the Fe3O4@C magnetic microspheres were synthesized by two-step reactions including solvothermal and hydrothermal reactions. Then, polyelectrolyte poly(diallyldimethylammonium chloride) (PDDA) was deposited onto the surface of Fe3O4@C magnetic microspheres by electrostatic adsorption. The presence of a layer of adsorbed positively charged PDDA on the Fe3O4@C magnetic microspheres ensures the efficient adsorption of negatively charged gold nanoparticles. Finally, Fe3O4@C@Au magnetic microspheres with core-shell structure were obtained by a selfassembly approach. To prove their practicability and great potential in bioapplications, the Fe3O4@C@Au magnetic microspheres were modified with 4-mercaptophenylboronic acid and applied to selective enrichment of glycoproteins and glycopeptides. 2. Experiment Section 2.1. Reagents and Materials. RNase B (RNB), horseradish peroxidase (HRP), myoglobin (MYO), β-casein from bovine milk, and 4-mercaptophenylboronic acid were purchased from Sigma Chemical (St. Louis, MO). All aqueous solutions were prepared with Milli-Q water by Milli-Q system (Millipore, Bedford, MA). All other chemicals and reagents were of the highest grade commercially available. 2.2. Sample Preparation. Bovine β-casein and HRP were each dissolved in 25 mM NH4HCO3 buffer at pH 8.3, and were treated with trypsin (2%, w/w) for more than 16 h at 37 °C to be completely digested, respectively. 2.3. Synthesis of Gold Nanoparticles. First, a 20 mL aqueous solution containing 0.25 mM HAuCl4 and 0.25 mM trisodium citrate was prepared (trisodium citrate acts as a capping agent and thus restricts particle growth). Next, 0.6 mL of a 0.01 M NaBH4 solution was added at once into the gold solution under constant stirring. Stirring was continued for another 30 s. The solution turned a wine red color indicating particle formation. The solution should be used within 24 h of its preparation. Beyond this time a thin gold film forms at the water surface, indicating particle aggregation.39 2.4. Synthesis of Fe3O4@C Magnetic Microspheres. The magnetic nanoparticels were synthesized through hydrothermal

reaction. Briefly, 1.35 g of FeCl3 · 6H2O was first dissolved in 75 mL of ethylene glycol under magnetic stirring. A clear yellow solution was obtained after stirring for 0.5 h. Then 3.60 g of sodium acetate was added to this solution. After being stirred for another 1 h, the resulting solution was transferred into a Teflon-lined stainless-steel autoclave with a capacity of 200 mL. The autoclave was sealed and heated at 200 °C for 16 h and cooled to room temperature. The black magnetic microspheres were collected with the help of a magnet field, followed by washing with recycled ethanol and deionized water six times. The product was then dried under vacuum at 50 °C for 12 h. In the next step, 0.2 g of magnetic microspheres was ultrasonicated for 10 min in 0.1 M HNO3, followed by washing with deionized water. Then, the treated Fe3O4 microspheres were redispersed in 0.5 M aqueous glucose solution and ultrasonicated for another10 min; next the suspension was transferred to autoclaves and kept at 180 °C for 6 h and cooled to room temperature. The Fe3O4@C microspheres were isolated with the help of a magnet and washed with deionized water six times. The Fe3O4@C microspheres were then dried under vacuum at 50 °C for 12 h. 2.5. Synthesis of Fe3O4@C@Au Magnetic Microspheres. The purified Fe3O4@C magnetic microspheres were functionalized with PDDA. Fe3O4@C magnetic microspheres were dispersed into an aqueous solution of 0.20% PDDA that contained 20 × 10-3 M Tris and 20 × 10-3 M NaCl and the resulting dispersion was stirred for 20 min. Residual PDDA was removed with the help of a magnet and the PDDA adsorbed microspheres were rinsed with water at least three times. The resulting magnetic microspheres (40 mg) were redispersed in 60 mL of the as-synthesized gold nanoparticles solution and the mixture was stirred for another 20 min. The supernatant was removed with the help of a magnet. The obtained Fe3O4@C@Au magnetic microspheres were washed with deionized water three times and dried under vacuum at 50 °C for 12 h. 2.6. Synthesis of Boronic Acid Functionalized Fe3O4@C@Au Magnetic Microspheres. The obtained Fe3O4@C@Au magnetic microspheres were resuspended in 20 mL of ethanol containing 20 mg of 4-mercaptophenylboronic acid and the solution was stirred for 4 h. The residual 4-mercaptophenylboronic acid was removed with the help of a magnet. The obtained boronic acid functionalized

Synthesis of Fe3O4@C@Au Magnetic Microspheres Fe3O4@C@Au magnetic microspheres were rinsed with ethanol three times and dried under vacuum at 50 °C for 12 h. The amount of 4-mercaptophenylboronic acid immobilized on the Fe3O4@C@Au magnetic microspheres was evaluated by HPLC. A 10 mg sample of magnetic microspheres was dispersed in 4 mL of 0.5 mg/mL 4-mercaptophenylboronic acid/ethanol solution for immobilization under gentle agitation, and 50 µL of supernatant was pipetted at certain time intervals from 0 min to 2 h. The supernatant was diluted four times with 50% methanol and centrifuged at 15000 g for 10 min. Then 10 µL of supernatant was submitted to HPLC analysis, using an Agilent 1100 LC system (Agilent Technologies, Waldbronn, Germany) and RP-HPLC column (Zorbax 300SB-C18, 4.6 × 250 mm, 5-Micron, Agilent Technologies, Waldbronn, Germany). The mobile phase contained 50% methanol and the flow rate was set at 0.8 mL/min. A 30 min isocratic elution step was adopted with UV detection wavelength at 254 nm. By analyzing the peak areas of parallel RPLC runs, the adsorption of 4-mercaptophenylboronic acid was estimated to be 50 µg/mg magnetic microspheres (Figure S3, Supporting Information). 2.7. Characterizations of Fe3O4@C@Au Magnetic Microspheres. Fourier-transform infrared (FT-IR) spectra were collected on a Nicolet Fourier spectrophotometer, using KBr pellets (USA). Transmission electron microscopy experiments were conducted on a JEOL 2011 microscope (Japan) operated at 200 kV. Powder X-ray diffraction (XRD) patterns were recorded on a Bruker D4 X-ray diffractometer with Ni-filtered Cu KR radiation (40 kV, 40 mA). The magnetic properties were characterized on a superconducting quantum interference device (SQUID) at 25 °C. 2.8. Enrichment of Glycoproteins and Glycopeptides with Boronic Acid Functionalized Microspheres. For glycoprotein capture, about 200 µg of microspheres was first washed extensively and dispersed in 50 mM NH4HCO3 solution (pH 8.0). Then 2 µL of RNase B (1 µg/µL) and myoglobin (1 µg/ µL) were added to the suspension. The capture procedure was carried out under gentle agitation at room temperature for 1 h. After reaction, the microspheres were washed several times with binding buffer and then incubated with 10 µL elution buffer containing 20% acetonitrile and 5% TFA at room temperature for 15 min. The final eluate was collected and dried in a SpeedVac for SDS-PAGE. For glycopeptides enrichment, the same procedure was adopted except that 100 µL of 5 ng/µL tryptic peptides solution generated from horseradish peroxidase was used for reaction. Finally, 2 µL of eluate and 2 µL of supernatant were spotted on a MALDI plate directly for mass spectrometry analysis. For selective enrichment of glycopeptides, a 2.5 ng/µL peptides solution of HRP was first mixed with the same volumes of tryptic β-casein peptides in the concentration ratio of 1:1, 1:5, and 1:10 (mol/mol), respectively, before being submitted to glycopeptide enrichment. 2.9. Mass Spectrometry. PMF analysis was performed by MALDI-QIT-TOFMS(AXIMAQIT;Shimadzu/Kratos,Manchester, UK). This instrument employs a three-dimensional ion trap with a time-of-flight mass measurement stage. Matrix-assisted laser desorption of peptides is produced by pulses of light (337 nm, 3-ns pulse width) generated by a nitrogen laser with a maximum pulse rate of 10 Hz. Each profile is composed of the accumulation of two laser shots. Ions are directed into the ion trap by means of an electrostatic lens assembly, designed to ensure high transmission for the MALDI beam into the ion trap and to minimize field strength directly above the sample surface.

J. Phys. Chem. C, Vol. 114, No. 20, 2010 9223 For QIT measurements, 2 µL of 12.5 mg/mL DHB dissolved in a 0.1% (v/v) TFA and 20% (v/v) acetonitrile solution was spotted on a MALDI sample target and dried. The measurements were carried out in positive ion mode. The Y-axis of MS spectra presented in the figures was expressed as the relative intensity of ion peaks, and in each spectrum the most intense ion peak was set to 100. The X-axis denotes the mass-to-charge ratio or m/z. 3. Results and Discussion The synthesis strategy is shown in the Scheme 1. The transmission electron microscope (TEM) reveals that the black Fe3O4 core was encapsulated into a thin gray shell (∼20 nm). The Fe3O4@C magnetic microspheres have a mean diameter of about 280 nm and a nearly spherical shape (Figure 1A). Fourier transform infrared (FTIR) was used to identify the functional groups present after hydrothermal reaction. In the FTIR spectrum, the characteristic peaks at 1699 and 1614 cm-1 correspond to CdO and CdC vibrations, respectively, which is a result of the aromatization of glucose. The bands at 1000-1300 cm-1 corresponding to the C-OH stretching and O-H bending vibrations reveal the presence of numerous hydrophilic groups and the incomplete carbonization of glucose (Figure S1, Supporting Information). The hydrophilic groups can endow Fe3O4@C microspheres with excellent aqueous dispersibility and stability. Moreover, as a result of the hydrothermal reaction, the surface of the Fe3O4@C magnetic microspheres turned to being negatively charged. The Fe3O4@C microspheres in aqueous solution (pH 7.0) had a negative ξ-potential of -59.3 mV. Next, in order to adsorb negatively charged Au nanoparticles onto the surface of the Fe3O4@C magnetic microspheres, we then modified them with PDDA, which provides a homogeneous distribution of positive charges. After the modification, the surface ξ-potential of the magnetic microspheres in aqueous solution (pH 7.0) was changed to +2.33 mV. This confirmed that the PDDA molecules were immobilized onto the magnetic microspheres. The Au nanoparticles were synthesized in aqueous solution through the reduction of HAuCl4 by NaBH4, and the solution turned a wine red color indicating Au nanoparticles formation (Figure 2A).39 As shown in Figure 2D, the solution turned transparent immediately when the PDDA modified Fe3O4@C magnetic microspheres were added to the Au nanoparticles’ dispersion, followed by applying a magnet. This suggests that the Au nanoparticles were adsorbed to the magnetic microspheres. The TEM image of the as-synthesized Fe3O4@C@Au magnetic microspheres was shown in Figure 1B. The mean diameter of the Au nanoparticles is estimated to be about 3 nm (Figure 1B, insert). It is clear that numerous dark nanodots were deposited onto the gray carbonaceous layer, which confirmed that the Fe3O4@C@Au magnetic microspheres have been successfully synthesized. The energy-dispersive X-ray analysis (EDXA) spectrum (Figure 1C) of the obtained Fe3O4@C@Au core-shell microspheres reveals the existence of Fe, Au, C, and O elements, further confirming the adsorption of Au nanoparticles on the Fe3O4@C microspheres. The wideangle X-ray diffraction (WAXRD) measurement result (Figure 3) also indicated that the Au nanoparticles were immobilized onto the surface of the magnetic microspheres. The magnetic characterization on a SQUID indicates that the obtained Fe3O4@C@Au microspheres possess superparamagnetism at room temperature and a magnetization saturation of 50.3 emu/ g. All the present results confirm that a typical core-shell Fe3O4@C@Au microsphere was observed.

9224

J. Phys. Chem. C, Vol. 114, No. 20, 2010

Qi et al.

Figure 1. TEM images of (A) Fe3O4@C magnetic microspheres and (B) Fe3O4@C@Au magnetic microspheres; (C) EDXA spectrum of the obtained Fe3O4@C@Au magnetic microspheres.

Figure 2. The procedure of self-assembling Au nanoparticles on PDDA-modified Fe3O4@C microspheres.

As mentioned above, the current synthesis strategies of boronic acid-functionalized magnetic microspheres were based on different chemical modifications.12-14 However, the method reported here was based on the robust interaction between the thiol group and gold nanoparticles. As the Experiment Section demonstrated, RP-HPLC was employed to measure the amount of 4-mercaptophenylboronic acid adsorbed on the Fe3O4@C@Au magnetic microspheres. The amount of immobilized 4-mercaptophenylboronic acid does not change distinctly after shaking for 1 h (Figure S2, Supporting Information), and it is estimated to be about 50 µg/mg Fe3O4@C@Au magnetic microspheres.

Figure 3. Wide-angle X-ray diffraction patterns of (a) Fe3O4@C microspheres and (b) Fe3O4@C@Au microspheres.

To investigate the enrichment efficiency and selectivity of the microspheres, we used tryptic peptides of a standard glycoprotein, horse radish peroxidase (HRP) for the enrichment experiments. There are theoretically eight glycopeptides contain-

Synthesis of Fe3O4@C@Au Magnetic Microspheres

Figure 4. Comparison of MALDI-TOF-MS spectrum of HRP. (A) The spectrum of supernatant after the boronic acid-functionalized magnetic microspheres treatment of 2.5 ng/µL tryptic digests of HRP. (B) The eluate after boronic acid-functionalized magnetic microspheres treatment of 2.5 ng/µL tryptic digests of HRP. Peptide fragments containing N-glycan moiety are marked with asterisks.

ing nine glycosylation sites in HRP which dominate the mass spectrum, and the masses of many observed glycopeptides species indicated a composition of a xylosylated, core-(R1-3)fucosylated trimannosyl N-glycan structure for the attached N-glycan moiety.40 Due to the specialty of the matrix-assisted laser desorption of quadrupole ion trap (QIT), there might be some fragment ions of glycopeptides in the MS map, therefore more than 8 glycopeptides signals are detected as shown in Figure 4B. Twelve [M + H]+ ions of glycopeptides were found in the eluate obtained from pretreatment of HRP peptides mixture adsorbed by 4-mercaptophenylboronic acid-functionalized magnetic microspheres. The signal at m/z 2533.7 was generated from the glycopeptide Ser295-Arg313 with the m/z at 3354.7, which contains a core-fucosylated and core-xylosylated trimannosyl N-glycan attached to N298. The mass decrement of 821 Da indicates the loss of glycan structure Man3GlcNAc1Xyl1. The glycosylated [M + H]+ ions at m/z of 1895.5, 2509.0, 3074.2, and 3749.4 are peptide fragments generated from the [M + H]+ ion signal at m/z 3895.4, which corresponded to the peptide L69-R92 with the same glycan structure as above attached to N87. The mass decrements among the m/z of 1895.5, 2509.7, and 3895.4 are due to the amino acids loss. Fucose loss causes the mass decrement of 146 Da between precursor ion and the m/z of 3749.4. The [M + H]+ ion signal at m/z 4984.8 corresponded to glycopeptide Leu214Arg236 having two glycosylated sites at N216 and N228. The signal at m/z 2592.1, which shows the strongest intensity in the QIT mass map, is a fragment generated from m/z 4984.8 containing one complete N-glycan moiety at N216. Compared to the mass map of the supernatant as shown in Figure 4A, although most glycopeptides are captured by magnetic microspheres, the [M + H]+ ion signal at m/z 2851.5, 3527.0, and 3673.0 cannot be effectively enriched. The ion at m/z 3527.0 is the fragment that lost a fucose of m/z 3673.0. Interestingly, the precursor ions at m/z 2851.5 and 3673.0 have the identical peptide moiety. The insufficient capture of these three glycopeptides might be caused by the specific peptide structure or by the suppression effect of other glycopeptides. Except for these three glycopeptides, almost all glycopeptides of HRP that have been reported are captured from the peptide mixture. Trace signals can be found in the supernatant solution after glycopeptides capture, which indicates our magnetic microspheres are able to remarkably separate glycopeptides from peptides mixture.

J. Phys. Chem. C, Vol. 114, No. 20, 2010 9225

Figure 5. SDS-PAGE of a mixture of model proteins without treatment and eluate after binding to the boronic acid-functionalized magnetic microspheres.

The selectivity of the magnetic microspheres was further evaluated by capturing glycosylated peptides from a complex peptides mixture of tryptic digests of HRP and β-casein with ratios of 1:1, 1:5, and 1:10 (mol/mol). Figure S3 (Supporting Information) shows the mass maps of the peptide mixture in different concentration ratios of HRP and β-casein, and the eluates after enrichment by magnetic microspheres. According to the mass spectra, the number of glycopeptides that can be detected is reduced intensively in peptide mixtures of two proteins. This might be due to the fact that the existence of nonglycosylated peptides can suppress the mass spectrometric response to glycopeptides, making it hard to identify the lowabundance glycopeptides. However, after the enrichment by 4-mercaptophenylboronic acid functionalized Fe3O4@C@Au magnetic microspheres, though there is relatively low peptide concentration (2.5 ng/µL) of HRP, several glycopeptides can be detected even when the concentration ratio of HRP and β-casein has reached 1:10. Compared to the previous approach based on boronic acid affinity probe,13,15 the proposed method we reported here shows comparable performance for the glycopeptides enrichment. As Zhou reported,13 the aminophenylboronic acid-functionalized magnetic nanoparticles show good enrichment performance when the peptide mixture was at a concentration of 5 ng/µL. However, our present experiment was conducted at a concentration of 2.5 ng/µL. It suggested that our method provides good enrichment efficiency. More recently, Tang and co-workers developed an on-plate-selective enrichment method for glycopeptides investigation.15 Their method shows great enrichment selectivity even when the concentration ratio of HRP and β-casein has reached 1:10. And our approach provides comparable enrichment performance. All the results demonstrated that our proposed method has good enrichment efficiency and good selectivity. Boronic acid modified Fe3O4@C@Au magnetic microspheres can be applied not only to glycopeptides enrichment, but also to the capture of glycoproteins. Here, a protein mixture of RNase B (RNB) containing high-mannose oligosaccharides (Man59GlcNAc2) structure41 and nonglycoprotein myoglobin (MYO) was employed for the capture of glycoprotein. As shown in Figure 5, two bands were observed indicating RNB and MYO in the protein mixture. However, only one band was observed in the eluates after enrichment by boronic acid modified Fe3O4@C@Au magnetic microspheres, which indicates the

9226

J. Phys. Chem. C, Vol. 114, No. 20, 2010

microspheres show efficient and specific enrichment of glycoproteins like RNB from complex samples. 4. Conclusion Herein, we present a novel and facilitated strategy to synthesize the core-shell structure Fe3O4@C@Au magnetic microspheres. Furthermore, the obtained microspheres were functionalized with 4-mercaptophenylboronic acid and used for glycopeptides and glycoproteins enrichment. Briefly, PDDA was deposited onto the surface of the as-prepared Fe3O4@C microspheres by electrostatic adsorption. As a result of the presence of the PDDA layer, the negatively charged Au nanoparticles can be efficiently anchored. The results presented in this article demonstrate that the core-shell structure Fe3O4@C@Au magnetic microspheres was successfully synthesized. The freshly synthesized Fe3O4@C@Au magnetic microspheres were modified with 4-mercaptophenylboronic acid for further glycopeptides/glycoproteins analysis. Our experiment data demonstrate that the boronic acid functionalized Fe3O4@C@Au magnetic microspheres show specific and efficient enrichment for glycoproteins and glycopeptides, which enable the microspheres to be applied in extensive glycolproteomics research in future work. Acknowledgment. This work was supported by the National Basic Research Priorities Program (Project 2007CB914100/3), the National Natural Science Foundation of China (Projects NSFC 20735005, NSFC20875017, and NSFC 30873132), the Technological Innovation Program of Shanghai (09JC1401100), and Shanghai Leading Academic Discipline Project (B109). Supporting Information Available: The FTIR spectrum of the Fe3O4@C magnetic microspheres; the absorbance of 4-mercaptophenylboronic acid on Fe3O4@C@Au magnetic microspheres at certain time intervals; and the MALDI-TOF-MS spectra of a peptides mixture of HRP and β-casein in the concentration ratios of (A) 1:1, (C) 1:5, and (E) 1:10 before enrichment and after enrichment. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Parodi, A. J. Annu. ReV. Biochem. 2000, 69, 69–93. (2) Lowe, J. B. Cell 2001, 104, 809–812. (3) Helenius, A.; Aebi, M. Science 2001, 291, 2364–2369. (4) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science 2001, 291, 2370–2376. (5) Roth, J. Chem. ReV. 2002, 102, 285–303. (6) O’Donnell, N. Biochim. Biophys. Acta 2002, 1573, 336–345. (7) Kaji, H.; Saito, H.; Yamauchi, Y.; Shinkawa, T.; Taoka, M.; Hirabayashi, J.; Kasai, K.; Takahashi, N.; Isobe, T. Nat. Biotechnol. 2003, 21, 667–672. (8) Zhang, H.; Li, X.; Martin, D. B.; Aebersold, R. Nat. Biotechnol. 2003, 21, 660–666.

Qi et al. (9) Hagglund, P.; Bunkenborg, J.; Elortza, F.; Jensen, O. N.; Roepstorff, P. J. Proteome Res. 2004, 3, 556–566. (10) Wada, Y.; Tajiri, M.; Yoshida, S. Anal. Chem. 2004, 76, 6560– 6565. (11) Miura, Y.; Hato, M.; Shinohara, Y.; Kuramoto, H.; Furukawa, J.; Kurogochi, M.; Shimaoka, H.; Tada, M.; Nakanishi, K.; Ozaki, M.; Todo, S.; Nishimura, S. Mol. Cell. Proteomics 2008, 7, 370–377. (12) Sparbier, K.; Wenzel, T.; Kostrzewa, M. J. Chromatogr., B 2006, 840, 29–36. (13) Zhou, W.; Yao, N.; Deng, C. Chem. Commun. 2008, 5577–5579. (14) Xu, Y.; Wu, Z.; Zhang, L.; Lu, H.; Yang, P.; Webley, P. A.; Zhao, D. Anal. Chem. 2009, 81, 503–508. (15) Tang, J.; Liu, Y.; Qi, D.; Yao, Y.; Deng, C.; Zhang, X. Proteomics 2009, 9, 5046–5055. (16) Frantzen, F.; Grimsrud, K.; Heggli, D. E.; Sundrehagen, E. J. Chromatogr., B 1995, 670, 37–45. (17) James, T. D.; Samankumara, K. R. A. Angew. Chem., Int. Ed. Engl. 1996, 35, 1910–1922. (18) Koyama, T.; Terauchi, K. J. Chromatogr., B 1996, 679, 31–40. (19) Lee, J. H.; Kim, Y. S.; Ha, M. Y.; Lee, E. K.; Choo, J. B. J. Am. Soc. Mass Spectrom. 2005, 16, 1456–1460. (20) Monzo, A.; Bonn, G. K.; Guttman, A. Anal. Bioanal. Chem. 2007, 389, 2097–2102. (21) Chen, H. M.; Deng, C. H.; Zhang, X. M. Angew. Chem. 2010, 49, 607–611. (22) Qi, D. W.; Lu, J.; Deng, C. H. J. Phys. Chem. C 2009, 113, 15854– 15861. (23) Chen, H. M.; Deng, C. H.; Li, Y.; Zhang, X. M. AdV. Mater. 2009, 21, 2200–2204. (24) Xu, X. Q.; Deng, C. H.; Gao, M. X.; Zhang, X. M. AdV. Mater. 2006, 18, 3289–3293. (25) Deng, Y. H.; Deng, C. H.; Qi, D. W.; Zhao, D. Y. AdV. Mater. 2009, 21, 1377–1382. (26) Penn, S. J.; He, L.; Natan, M. J. Curr. Opin. Chem. Biol. 2003, 7, 609–615. (27) You, C.; Miranda, O. R.; Gider, B.; Ghosh, P. S.; Kim, I.; Erdogan, B.; Krovi, S. A.; Bunz, U. H. F.; Rotello, V. M. Nat. Nanotechnol. 2007, 2, 318–323. (28) Liu, H. L.; Sonn, C. H.; Wu, J. H.; Lee, K.-M.; Kim, Y. K. Biomaterials 2008, 29, 4003–4011. (29) Kouassi, G. K.; Irudayaraj, J. Anal. Chem. 2006, 78, 3234–3241. (30) Yao, G.; Zhang, H.; Deng, C.; Lu, H.; Zhang, X.; Yang, P. Rapid Commun. Mass Spectrom. 2009, 23, 3493–3500. (31) Cui, Y.; Hui, W.; Wang, H.; Chen, C. Sci. China, Ser. B: Chem. 2004, 47, 152–158. (32) Stoeva, S. I.; Huo, F.; Lee, J.; Mirkin, C. A. J. Am. Chem. Soc. 2005, 127, 15362–15363. (33) Lyon, J. L.; Fleming, D. A.; Stone, M. B.; Schiffer, P.; Williams, M. E. Nano Lett. 2004, 4, 719–723. (34) Yu, H.; Chen, M.; Rice, P.; Wang, S.; White, R.; Sun, S. Nano Lett. 2005, 5, 379–382. (35) Wang, Y.; Angelatos, A. S.; Caruso, F. Chem. Mater. 2008, 20, 848–858. (36) Wang, D. Y.; Rogach, A. L.; Caruso, F. Nano Lett. 2002, 2, 857– 861. (37) Caruso, F.; Schu¨ler, C. Langmuir 2000, 16, 9595–9603. (38) Johnston, A. P. R.; Cortez, C.; Angelatos, A. S.; Caruso, F. Curr. Opin. Colloid Interface Sci. 2006, 11, 203–209. (39) Jana, N. R.; Gearheart, L.; Murphy, C. J. AdV. Mater. 2001, 13, 1389–1393. (40) Wuhrer, M.; Hokke, C. H.; Deelder, A. M. Rapid Commun. Mass Spectrom. 2004, 1741–1748. (41) Pitchaywasin, S.; Isobe, M. Biosci., Biotechnol., Biochem. 2004, 68, 1424–1433.

JP9114404