Magnetic MSP@ZrO2 Microspheres with Yolk–Shell Structure

May 16, 2014 - First, a large amount of the generated hydrolyzate Zr(OH)4 was firmly fixed onto the surface of the cross-linked polymethylacrylic acid...
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Magnetic MSP@ZrO2 Microspheres with Yolk−Shell Structure: Designed Synthesis and Application in Highly Selective Enrichment of Phosphopeptides Wan-Fu Ma,†,§ Cheng Zhang,‡,§ Yu-Ting Zhang,† Meng Yu,† Jia Guo,† Ying Zhang,‡ Hao-Jie Lu,*,‡ and Chang-Chun Wang*,† †

State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai 200433, China ‡ Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China S Supporting Information *

ABSTRACT: Magnetic yolk−shell MSP@ZrO2 microspheres consisting of a movable magnetic supraparticle (MSP) core and a crystalline ZrO2 shell were synthesized via a two-step controlled “sol−gel” approach for the first time. First, a large amount of the generated hydrolyzate Zr(OH)4 was firmly fixed onto the surface of the cross-linked polymethylacrylic acid matrix via a strong hydrogen-bonding interaction between Zr(OH)4 and the carboxyl groups. Then a calcination process was adopted to convert the Zr(OH)4 into a continuous ZrO2 shell and simultaneously make the ZrO2 shell crystallized. At the same time, the polymer matrix could be selectively removed to form a yolk−shell structure, which has better dispersibility and higher adsorbing efficiency of phosphopeptides than its solid counterpart. The formation mechanism of such yolk−shell microspheres could be reasonably proved by the results of TEM, TGA, VSM, XRD, and FT-IR characterization. By taking advantage of the unique properties, the yolk−shell MSP@ZrO2 exhibited high specificity and great capability in selective enrichment of phosphopeptides, and a total of 33 unique phosphopeptides mapped to 33 different phosphoproteins had been identified from 1 mL of human saliva. This result clearly demonstrated that the yolk−shell MSP@ZrO2 has great performance in purifying and identifying the low-abundant phosphopeptides from real complex biological samples. Moreover, the synthetic method can be used to produce hybrid yolk− shell MSP@ZrO2−TiO2. oxide@silica,34 and so on. However, to the best of our knowledge, very few ZrO2-based YSNs have been constructed and no attempt has been made to integrate a magnetic core with a ZrO2 shell to produce magnetic ZrO2 YSNs due to a lack of effective synthetic strategy. Therefore, a method to fabricate well-defined magnetic ZrO2 YSNs is still challenging and highly desired. Reversible protein phosphorylation, as one of the most important protein PTMs, plays a vital role in regulating many complex biological processes, such as cellular growth and division, and signaling transduction.35−37 Besides, many diseases (e.g., cancer and Alzheimer) are relevant to abnormal phosphorylation, and some phosphopeptides can be used as the biomarker for medical inspection.38−40 However, it is still a great challenge to directly detect phosphopeptides due to their low dynamic stoichiometry and signal suppression of nonphosphorylated peptides. Thus, it is necessary to selectively

1. INTRODUCTION In recent years, much scientific effort has been made to design and fabricate multifunctional nanomaterials with controlled morphologies, tailored structures, and desired functionalities.1−7 As an important member of hollow-structured nanoarchitecture, yolk−shell nanoparticles (YSNs) or so-called “nanorattles” with a distinctive core@void@shell configuration have gained immense interest because of their unique property and potential applications.8−19 The freely movable core, the tunable functional shell, together with the intermediate hollow space can offer YSNs with new properties, thus rendering them attractive for applications in drug delivery,10,11 nanoreactors,12−18 lithium-ion batteries,19,20 and so on. To date, many approaches have been developed to prepare YSNs, such as selective etching or dissolution method,12,14,17 soft template,13,22,23 ship in bottle,24 Ostwald ripening,25,26 galvanic replacement,27,28 and methods based on the Kirkendall effect.29 On the basis of these techniques, a series of YSNs covering a wide range of chemical compositions have been fabricated, including metal−NPs@polymer, 30 metal−NPs@silica, 31 metal−NPs@carbon,32 metal−NPs@metal−oxides,33 metal− © 2014 American Chemical Society

Received: April 10, 2014 Revised: May 13, 2014 Published: May 16, 2014 6602

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diffuse reflectance spectra were scanned over the range of 400−4000 cm−1. Thermogravimetric analysis (TGA) measurements were performed on a Pyris 1 TGA instrument. All measurements were taken under a constant flow of nitrogen of 40 mL/min. The temperature was first increased from room temperature to 100 °C and held until constant weight and then increased from 100 to 800 °C at a rate of 20 °C/min. Magnetic characterization was carried out with a vibrating sample magnetometer (VSM) on a model 6000 physical property measurement system (Quantum, USA) at 300 K. XRD patterns were collected on a X’Pert Pro (Panalytical, The Netherlands) diffraction meter with Cu KR radiation at λ = 0.154 nm operating at 40 kV and 40 mA. 2.2. Synthesis of MSP@PMAA Core/Shell Microspheres. MSP@PMAA core/shell microspheres were prepared similar to our prior work.45 Magnetic supraparticles (MSPs) were prepared through a modified solvothermal reaction. Typically, 1.350 g of FeCl3·6H2O, 3.854 g of NH4Ac, and 0.4 g of sodium citrate were dissolved in 70 mL of ethylene glycol. The mixture was stirred vigorously for 1 h at 170 °C to form a homogeneous black solution and then transferred into a Teflon-lined stainless-steel autoclave (100 mL capacity). The autoclave was heated at 200 °C and maintained for 16 h, and then it was cooled to room temperature. The black product was washed with ethanol and collected with the help of a magnet. The cycle of washing and magnetic separation was repeated several times. The final product was dispersed in ethanol for further use. Modification of MSPs with MPS was achieved by adding 40 mL of ethanol, 10 mL of deionized water, 1.5 mL of NH3·H2O, and 0.3 g of MPS into the MSPs ethanol suspension and vigorously stirring the mixture for 24 h at 60 °C. The obtained product was separated using a magnet and washed with ethanol to remove excess MPS. The resultant MSP@MPS nanoparticles were dried in a vacuum oven at 40 °C until constant weight. Coating a PMAA layer onto MSP@MPS nanoparticles was executed by reflux−precipitation polymerization46 of MAA, with MBA as the cross-linker and AIBN as the initiator, in acetonitrile. Typically, about 200 mg of MSP@MPS seed nanoparticles was dispersed in 160 mL of acetonitrile in a dried 250 mL single-necked flask with the aid of ultrasonic. Then a mixture of 1.2 mL of MAA, 300 mg of MBA, and 30 mg of AIBN was added to the flask to initiate polymerization. The flask submerged in a heating oil bath was attached with a Liebig condenser. The reaction mixture was heated from ambient temperature to the boiling state within 30 min, and the reaction was ended after 2 h. The obtained MSP@PMAA microspheres were collected by magnetic separation and washed with ethanol in order to eliminate excess reactants and few generated polymer microspheres. 2.3. Synthesis of MSP@PMAA@PMAA−Zr(OH)4 Core/Shell/ Shell Microspheres. MSP@PMAA@PMAA−Zr(OH)4 core/shell/ shell microspheres were synthesized by adsorbing generated hydrolyzate Zr(OH)4 onto the surface of MSP@PMAA in ethanol solvent at 60 °C. Zr(OH)4 was generated by hydrolyzing Zr(OBu)4 in the presence of a very small amount of deionized water. Briefly, about 100 mg of the as-prepared MSP@PMAA was dispersed in 100 mL of ethanol containing 500 μL of deionized water at 60 °C. Finally, 250 μL of Zr(OBu)4 was dissolved in 20 mL of ethanol, and the solution was added to the above suspension under stirring. After reacting for 3 h, the products were collected by magnetic separation and washed with ethanol several times. 2.4. Synthesis of MSP@PMAA@PMAA−Zr(OH)4−Ti(OH)4 Core/Shell/Shell Microspheres. The procedures for preparation of MSP@PMAA@PMAA−Zr(OH)4−Ti(OH)4 core/shell/shell microspheres were similar to that of MSP@PMAA@PMAA−Zr(OH)4 except for changing the Zr(OBu)4 to a combination of Zr(OBu)4 and Ti(OBu)4. 2.5. Synthesis of MSP@ZrO2 Yolk−Shell Microspheres. The yolk−shell MSP@ZrO2 microspheres were achieved by calcining the obtained MSP@PMAA@PMAA-Zr(OH)4 microspheres at 500 °C under an atmosphere of air. Typically, 20 mg of MSP@PMAA@ PMAA−Zr(OH)4 was dispersed in 2 mL of ethanol in the combustion boat and then dried for 10 min in the vacuum drying oven until a layer

enrich phosphopeptides from the complicated digestion in phosphoproteome analysis. Immobilized metal affinity chromatography (IMAC) and metal oxide/hydroxide affinity chromatography (MOAC) are two commonly used enrichment strategies. In the MOAC method, phosphopeptides can be firmly adsorbed onto the surface of the metal oxides, such as TiO2,41 ZrO2,42 and Fe2O343 in acidic environment via the bridging bidentate interaction between the phosphate groups and the surface of the metal oxides while being easily desorbed from the surface in the alkaline condition.44 In comparison to TiO2-based material, much less work could realize high-quality ZrO2 nanomaterial, especially a magnetic ZrO2 nanocomposite with high performance. The main reason is that the hydrolysis and condensation of zirconium precursor is much more difficult to be controlled than that of titanium precursor. The aim of this work is to develop a novel method that could well control the hydrolysis and condensation process in order to synthesize uniform yolk−shell magnetic ZrO2 microspheres and then investigate their performance in selective enrichment of phosphopeptides from complicated samples. Herein, for the first time, high-quality yolk−shell MSP@ZrO2 microspheres have been rationally designed and synthesized via a two-step controlled “sol−gel” process. The formation mechanism was proposed and demonstrated by monitoring the synthetic process with TEM, TGA, VSM, XRD, and FT-IR characterization. The as-synthesized MSP@ZrO2 possesses an eye-like morphology, hierarchical yolk−shell structure, highly crystalline ZrO2 shell, and high magnetic susceptibility. The performance of the MSP@ZrO2 in selective enrichment of phosphopeptides is evaluated from both lab-made and real biological samples. The application effect indicates that the MSP@ZrO2 has high specificity and great capability in selective enrichment of lowabundant phosphopeptides from complicated biological samples. Moreover, this synthetic route is also proved to be applicable to fabricate hybrid yolk−shell MSP@ZrO2−TiO2.

2. EXPERIMENTAL SECTION 2.1. Materials. Iron(III) chloride hexahydrate (FeCl3·6H2O), ammonium acetate (NH4Ac), ethylene glycol (EG), anhydrous ethanol, trisodium citrate dihydrate, aqueous ammonia solution (25%), and methylacrylic acid (MAA) were purchased from Shanghai Chemical Reagents Co. and used as received. γ-Methacryloxypropyltrimethoxysilane (MPS) was obtained from Jiangsu Chen Guang Silane Coupling Reagent Co., Ltd. N,N′-Methylenebis(acrylamide) (MBA) was bought from Fluka and recrystallized from acetone. 2,2Azobis(isobutyronitrile) (AIBN) was supplied by Sinopharm Chemical Reagents Co. Zirconium n-butoxide (Zr(OBu)4) was available form TCI and is analytical grade. Tetrabutyl orthotitanate (TBOT) was bought from Jiangsu Qiang Sheng Chemical Reagent Co., Ltd. βCasein, bovine serum albumin (BSA, 95%), 2,5-dihydroxybenzoic acid (2,5-DHB, 98%), ammonium bicarbonate (ABC, 99.5%), and 1−1(tosylamido)-2-phenyl-ethyl chloromethyl ketone (TPCK)-treated trypsin (E.G 2.4.21.4) were purchased from Sigma (St.Louis, MO). Acetonitrile (ACN, 99.9%) and trifluoroacetic acid (TFA, 99.8%) were purchased from Merck (Darmstadt, Germany). Phosphoric acid (85%) was purchased from Shanghai Feida Chemical Reagents Ltd. (Shanghai, China). Matrix DHB was dissolved in acetonitrile (ACN)/water (50/50, v/v) solution containing 1% H3PO4 by keeping DHB at 10 mg/mL. Deionized water (18.4 MΩ cm) used for all experiments was obtained from a Milli-Q system (Millipore, Bedford, MA). Field-emission transmission electron microscopy (FE-TEM) images were taken on a JEM-2100F transmission electron microscope at an accelerating voltage of 200 kV. Samples dispersed at an appropriate concentration were cast onto a carbon-coated copper grid. Fourier transform infrared spectra (FT-IR) were determined on a NEXUS-470 FT-IR spectrometer over a potassium bromide pellet, and 6603

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Scheme 1. Schematic Illustration of the Synthetic Procedure for Preparation of Yolk−Shell MSP@ZrO2 Microspheres

μL) was deposited for MS analysis. MALDI-TOF mass spectrometry analysis was performed in positive reflection mode on a 5800 Proteomic Analyzer (Applied Biosystems, Framingham, MA) with a Nd: YAG laser at 355 nm, a repetition rate of 200 Hz, and an acceleration voltage of 20 kV. The range of laser energy was optimized to obtain good resolution and signal-to-noise ratio (S/N) and kept constant for further analysis. External mass calibration was performed using standard peptides from myoglobin digests. 2.11. 1D Nanoflow Liquid Chromatography-Tandem MS (LC-MS/MS) Analysis. Phosphopeptides enriched from a tryptic digest of saliva were divided into 3 parts, and each part was analyzed by 1D nanoflow LC-MS/MS. Liquid chromatography was performed on a nano Acquity UPLC system (Waters Corp., Milford, CT) connected to a LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with an online nanoelectrospray ion source (Michrom Bioresources, Auburn, CA). Peptides were resuspended with solvent A (2% acetonitrile, 0.1% formic acid in water, 25 μL). Peptide solution (20 μL) was loaded onto the Captrap Peptide column (2 mm × 0.5 mm, Michrom Bioresources, Auburn, CA) at a 20 μL min−1 flow rate of solvent A for 5 min and then separated on a Magic C18AQ reverse phase column (100 μm id ×15 cm, Michrom Bioresources, Auburn, CA) with a linear gradient: starting from 5% B (90% acetonitrile, 0.1% formic acid in water) to 45% B (in other words, from 95% A to 55% A, the same below) in 100 min. The column flow rate was maintained at 500 nL min−1. The electrospray voltage (1.6 kV) versus the inlet of the mass spectrometer was used. A LTQ Orbitrap XL mass spectrometer was operated in data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra with one microscan (m/z 350−1800) was acquired in the Obitrap with a mass resolution of 60 000 at m/z 400, followed by MS/MS of the eight most intense peptide ions in the LTQ analyzer. The automatic gain control (AGC) was set to 106 ions, with maximum accumulation times of 500 ms. A single charge state was rejected, and dynamic exclusion was used with two microscans in 15 and 30 s exclusion duration. For MS/MS, precursor ions were activated using 35% normalized collision energy at the default activation q of 0.25 and an activation time of 30 ms. The mass spectrometer was set so that one full MS scan was followed by three MS2 scans and three neutral loss MS3 scans. Detection of phosphopeptides was performed in which the mass spectrometer was set as a full scan MS followed by three data-dependent MS2. Subsequently, the MS3 spectrum was automatically triggered when the three most intense peaks from the MS2 spectrum corresponded to a neutral loss event of 98, 49, and 32.67 ± 1 Da for the precursor ion with 1+, 2+, and 3+ charge states, respectively. Spectra were recorded with Xcalibur (version 2.0.7) software. 2.12. Data Processing and Analysis. All MS/MS spectra in raw files were converted to single *.mgf files using MassMatrix Mass

of membrane formed. Afterward, the boat was put into the muffle furnace, heated slowly to 500 °C, and calcined for 3 h. Then it was cooled to room temperature. The resulting yolk−shell MSP@ZrO2 microspheres were washed several times with ethanol and then redispersed in ethanol for subsequent use. 2.6. Synthesis of Yolk−Shell MSP@ZrO2−TiO2 Microspheres. Procedures for preparation of yolk−shell MSP@ZrO2−TiO2 microspheres were similar to that of MSP@ZrO2 except for changing MSP@PMAA@PMAA−Zr(OH)4 to MSP@PMAA@PMAA−Zr(OH)4−Ti(OH)4. 2.7. Preparation of Tryptic Digest of Standard Proteins. βCasein and BSA were each dissolved in 25 mM ABC at pH 8.0 (1 mg/ mL for each protein) and denatured by boiling for 10 min. Protein solutions were then incubated with trypsin at an enzyme/substrate ratio of 1:40 (w/w) for 12 h at 37 °C to produce proteolytic digests. The tryptic peptide mixtures were stored at −20 °C until further use. 2.8. Preparation of Tryptic Digest of Proteins Extracted from Saliva. A 1 mL amount of saliva was collected from a healthy volunteer at 9:30 a.m. who had refrained from eating and drinking. The collection was placed at 0 °C and added with 20 μL of phosphatase inhibitors (1 mM Na3VO4, 1 mM NaF). The mixture was then centrifuged twice at 12 000g for 5 min each time. The supernatant was lyophilized and saved for further use. The lyophilized sample (about 1 mg) was then added with 120 μL of urea denaturing solution (8 M urea, 0.4 M ABC) and treated with 100 mM DTT (to obtain a final concentration of 10 mM) for 1 h at 37 °C. Samples were further alkylated with 100 mM IAA (to obtain a final concentration of 30 mM) at 37 °C for 30 min in the dark and treated with additional 100 mM DTT (to obtain a total amount of DTT that was one-half that of IAA) for 30 min at 37 °C. Prior to adding trypsin, the solution of the protein was diluted to 2 mL with 25 mM ABC buffer. This protein solution was incubated for 12 h with 25 μg of trypsin and another 4 h with 25 μg of trypsin at 37 °C for proteolysis. This tryptic digest of saliva was lyophilized and saved for further use. 2.9. Selective Enrichment of Phosphopeptides with MSP@ ZrO2 Yolk−Shell Microspheres. The obtained MSP@ZrO2 was first washed with ethanol three times and then suspended in deionized water at 1 mg/mL. Tryptic digest of β-casein and BSA or a protein mixture from saliva was dissolved in 500 μL of loading buffer (80% ACN containing 5% TFA); then 10 μL of MSP@ZrO2 was added and incubated at room temperature for 30 min. Afterward, MSP@ZrO2 with captured phosphopeptides was separated from the mixed solutions by applying an external magnet. After washing with 100 μL of loading buffer twice to remove the nonspecifically adsorbed peptides, the trapped phosphopeptides were eluted with 50 μL of 10% NH3·H2O for further MS analysis. 2.10. MALDI Mass Spectrometry. One microliter of the eluate was deposited on the MALDI probe, and then matrix solution DHB (1 6604

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Figure 1. Representative TEM images of (a) MSPs, (b) MSP@PMAA, (c) MSP@PMAA@PMAA−Zr(OH)4, and (d) yolk−shell MSP@ZrO2 (all scale bars are 100 nm).

Figure 2. Representative TEM images of MSP@PMAA@PMAA−Zr(OH)4 prepared (a) without water and (b) with water at a molar ratio of about 50 to Zr(OBu)4 (all scale bars are 100 nm). Spectrometric Data File Conversion Tools (version 3.9, http://www. massmatrix.net/download). The *.mgf files were searched using the Mascot Daemon software (Version 2.3.0, Matrix Science, London, U.K.) based on the Mascot algorithm. The database used to search was the UniProtKB/Swiss-Prot database (Taxonomy: vipera; release 2013_04_25, with 142 entries). The searching parameters were set up as follows: Peptides were searched using trypsin specified. A maximum of two missed cleavages (MCs) was allowed. Carbamidomethylation on cysteine (57.02146 Da) was set as fixed modifications. In addition, oxidation on methionine (15.9949 Da) was set as variable modifications. The peptide mass tolerance was 20 ppm, and the fragment ion tolerance was 1.0 Da. Peptide identifications were considered for expectation values lower than 0.05 (p < 0.05). The expectation cutoff value of 0.05 was applied in the MASCOT ion score to avoid peptide identifications out of the 95% confidence interval to be selected.

the surface of MSPs for facilitating encapsulation of the robust PMAA layer by reflux−precipitation polymerization (RPP).46,47 Subsequently, a considerable amount of Zr(OH)4 was firmly fixed onto the surface of the PMAA layer with the aid of the strong hydrogen-bond interaction between Zr(OH)4 and the carboxyl groups. Finally, the MSP@PMAA@PMAA−Zr(OH)4 microspheres were subjected to a calcination process for killing three birds with one stone: polycondensation to form dense ZrO2 shell, crystallization of the ZrO2 shell, and removal of the organic component to shape into the yolk−shell structure. Representative TEM images of MSPs and MSP@PMAA core/shell microspheres are shown in Figure 1a and 1b. The MSPs gave an average diameter of ca. 200 nm and were uniform both in shape and in size. After coating with PMAA layer, the obtained MSP@PMAA microspheres possessed a distinctive core/shell structure and the size of the composite microspheres increased to around 270 nm. The sacrificial PMAA layer played a key role for formation of target rattle-type MSP@ZrO2 microspheres. On one hand, the cross-linked matrix with abundant carboxyl groups provided a scaffold for anchoring of the hydrolyzed Zr(OH)4 species as well as the followed condensation process. On the other hand, this interim sacrificial layer could be easily removed during the calcination process. As seen in Figure 1c, the Zr(OH)4 species generated

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Yolk−Shell MSP@ZrO2 Microspheres. The overall synthetic route employed for preparation of hierarchical yolk−shell MSP@ ZrO2 microspheres is schematically illustrated in Scheme 1. Briefly, MSPs stabilized by sodium citrate were first synthesized via a modified solvothermal reaction. Then the as-prepared MSPs were modified by MPS to form active double bonds on 6605

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Figure 3. (a) FT-IR spectra and (b) X-ray diffraction patterns of (i) MSP@MPS, (ii) MSP@PMAA, (iii) MSP@PMAA@PMAA−Zr(OH)4, and (iv) MSP@ZrO2.

Figure 4. (a) TGA curves and (b) magnetic hysteresis curves (inset, magnified scale of the plot) of (i) MSP@MPS, (ii) MSP@PMAA, (iii) MSP@ PMAA@PMAA−Zr(OH)4 and (iv) yolk−shell MSP@ZrO2.

In addition, too much water will lead to generation of a large amount of secondary nanoparticles and almost no anchoring of Zr(OH)4 on the surface of MSP@PMAA. The influence of water amount on formation of a uniform PMAA−Zr(OH)4 shell could be explained by the effect of water on the hydrolysis of the precursor and nucleation rate of the generated Zr(OH)4 in ethanol solvent. Only if a small amount of water is used, the hydrolyzed Zr(OH)4 has a weak tendency to nucleate. The optimized molar ratio of water to Zr(OBu)4 is around 50. The whole fabrication process was continued to be vigorously investigated by FT-IR spectroscopy and powder Xray diffraction. In comparison to MSP@MPS, the new peaks appearing at 1708 and 1527 cm−1 (Figure 3a) attributed to the stretching vibration of CO of carboxyl groups and the bending vibration of N−H of MBA proved the presence of the PMAA layer in MSP@PMAA. After absorbing the Zr(OH)4 species it could be clearly observed that the intensity of the peak at 1708 cm−1 was dramatically decreased, which should be caused by the interaction between Zr(OH)4 and the carboxyl groups. As the PMAA layer was removed during the calcination process, the corresponding signals disappeared in the spectrum of yolk−shell MSP@ZrO2. The crystallization transition before and after calcination was further studied by powder X-ray diffraction. As shown in Figure 3b, prior to the calcination process, MSPs, MSP@PMAA ,and MSP@PMAA@PMAA− Zr(OH)4 all showed a simple PXRD pattern, which is well ascribed to the typical cubic structure of Fe3O4 (JCPDS 19-

through hydrolyzing the precursor of Zr(OBu)4 with only a very small amount of ionized water were rigidly fixed onto the external surface of MSP@ZrO2 to construct a core/shell/shell structure. Then the anchored Zr(OH)4 could react with their neighboring counterparts to constitute a continuous ZrO2 shell. In the meantime, the interim PMAA shell was completely eliminated and a well-defined yolk−shell structure composed of a movable MSP core and a ZrO2 outer shell was successfully obtained (Figure 1d). In addition, the selected-area electron diffraction (SAED) patterns recorded from a certain area of the PMAA−Zr(OH)4 or ZrO2 shell revealed that Zr(OH)4 was amorphous (Figure 1c, inset) while the ZrO2 shell is polycrystalline (Figure 1d, inset). The feeding amount of the ionized water is critical for obtaining high-quality covering of the Zr(OH)4 species onto the surface of MSP@PMAA. Without adding any water, the precursor Zr(OBu)4 could not be transformed into Zr(OH)4 via hydrolysis and the interaction between Zr(OBu)4 and PMAA layer is too poor to ensure Zr(OBu)4 adsorbs onto the surface of the microspheres (Figure 2a). Only in the presence of water the precursor Zr(OBu)4 could be hydrolyzed into Zr(OH)4 in order to obtain a satisfactory covering of ZrO2. However, the dosage of water must be strictly restricted to a very small amount. When relatively more water is added into the system, obvious secondary nanoparticles were formed and consequently influenced the adsorption efficiency of Zr(OH)4 species as well as the morphology of the resulted microspheres. 6606

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Scheme 2. Schematic Illustration of the Typical Process for Selective Enrichment of Phosphopeptides Using Yolk−Shell MSP@ ZrO2 and Interaction between Yolk−Shell MSP@ZrO2 and Phosphopeptides

significant increase from 14.6 to 29.9 emu/g, indicating the content of the MSP core is about 36.4 wt % while the remainding 63.6 wt % is corresponding to the content of ZrO2. Thanks to the high Ms value of yolk−shell MSP@ZrO2, the final product could be separated from the dispersion within only 10 s when an external magnetic field was applied. 3.2. Evaluation of Phosphopeptides Enrichment Capabilities of the Yolk−Shell MSP@ZrO2 Microspheres. Phosphopeptides can be captured by ZrO2 through bridging bidentate bonds between the phosphoric acid groups of phosphopeptides and ZrO2 at acidic condition while being desorbed from the surface of ZrO2 at alkaline environment (Scheme 2). Therefore, the “catch and release” process of phosphopeptides could be easily realized by adjusting the pH of the solution. To investigate the capabilities of the obtained yolk−shell MSP@ZrO2 for selective enrichment of phosphopeptides, bovine β-casein was selected as the standard phosphoprotein due to its well-characterized phosphorylated sites.48 To test the specificity of yolk−shell MSP@ZrO2 in phosphopeptides enrichment, tryptic digest of β-casein was mixed with digest of standard nonphosphoprotein BSA at a molar ratio of 1:100. The standard phosphoprotein β-casein would generate three phosphopeptides after trypsin digestion, including m/z at 2061.83, 2556.09, and 3122.27 in the MALDI spectrum. In a typical enrichment procedure, the β-casein digest and BSA digest were first dissolved in the loading buffer consisting of 80% acetonitrile and 5% trifluoroacetic acid (TFA). Then the solution was incubated with yolk−shell MSP@ZrO2. The yolk−shell MSP@ZrO2 with captured phosphopeptides were separated from the mixture by applying an external magnetic field and washing with the loading buffer several times to remove nonspecifically adsorbed peptides. Finally, the phosphopeptides were eluted from MSP@ZrO2 with 10% NH3·H2O and used for MALDI-TOF MS analysis. Before enrichment, the spectrum is dominated by nonphosphopeptides without detection of any phosphopeptides (Figure 5a). After selective enrichment, signals of the three phosphopeptides could be easily detected with a clean background, shown in Figure 5b. This result confirmed the high enrichment selectivity of yolk−shell MSP@ZrO2 toward phosphopeptides. The high selectivity is attibuted to the highly pure and crystalline ZrO2 surface as well as the yolk−shell structure. The relatively lower mass density of yolk−shell structure makes the yolk−shell MSP@ZrO2 have better dispersibility than its solid counterpart,42 which could help to improve the enrichment selectivity. To our knowledge, the concentration of biological active peptides is always at an extremely low level. Therefore, the enrichment sensitivity of the MSP@ZrO2 was investigated, as illustrated in the MALDI mass spectrum shown in Figure 5c. The targeted three phosphopeptides could be easily enriched and detected at a signal-to-noise ratio of 82, 294, and 74 for the phosphopeptide with m/z of 2061.83, 2556.09, and 3122.27, respectively, even when the

629). However, after calcination at air atmosphere for 3 h, the PXRD pattern for the synthesized yolk−shell MSP@ZrO2 microspheres was noticeably different from the former patterns; six distinct XRD peaks were clearly observed at 2θ values of 30.3°, 35.3°, 50.4°, 60.3°, 63.0°, and 74.6° (marked with “T” in the spectrum), which are well assigned to the (101), (002), (112), (211), (202), and (220) reflection planes of the bodycentered tetragonal phase of ZrO2 (t-ZrO2) crystals (JCPDS card No. 79-1771). This result agrees well with that of SAED characterization. Besides the crystallization transition of the ZrO2 shell, the Fe3O4 component in the MSP core also transformed to γ-Fe2O3 during the calcination process under air.43 To quantitatively determinate the composition of these composite microspheres, thermogravimetric analysis (TGA) was executed (Figure 4a). As the organic components decomposed at high temperature while the inorganic components remained, the TGA curves of MSP@MPS and MSP@PMAA show the Fe3O4 weight percentage of these two microspheres. A weight loss of 17.8 wt % MSP@MPS is attributed to the weight ratio of the citrate stabilizer and a small amount of MPS, indicating the Fe3O4 content is 82.2 wt %. After the coating by the PMAA layer, the Fe3O4 content in composite microspheres dramatically declined to 42.4 wt %. When Zr(OH)4 was introduced to the system, it is more difficult to directly know the Fe3O4 content of the microspheres because Zr(OH)4 will be converted into ZrO2 during the TGA measurement, and this transformation will also lead to some additional weight loss. Through calculation the Fe3O4 content is about 18.1% in weight, while the content of Zr(OH)4 can reach up to 57.2 wt %. Since all of the organic component of MSP@PMAA@PMAA−Zr(OH)4 was removed and Zr(OH)4 was transformed into ZrO2 during the calcination process, yolk−shell MSP@ZrO2 nearly has no weight loss in the TGA curve. The magnetic properties of the four kinds of microspheres were studied using a vibrating sample magnetometer (VSM) (Figure 4b). No obvious magnetic hysteresis loops (Hc < 30 Oe) were observed for all three kinds of microspheres from the field-dependent magnetization plots in the inset of Figure 4b, which indicated that they all possessed a superparamagnetic feature at room temperature. The superparamagnetism is coming from the small nanocrystals in the MSP cores, which behave as superparamagnets. As a control, the saturation magnetization (Ms) value of the MSPs was measured; it reached 67.2 emu/g. Upon coating of the PMAA and adsorbing of Zr(OH)4, the Ms values for MSP@PMAA and MSP@ PMAA@PMAA−Zr(OH)4 were reduced to 35.5 and 14.6 emu/g. Accordingly, the Fe3O4 content of these two composite microspheres was estimated to be 43.3 and 17.8 wt %, respectively, which agrees well with the TGA results and offers powerful evidence of the existence of Zr(OH)4 instead of ZrO2 in the MSP@PMAA@PMAA−Zr(OH)4. Moreover, after elimination of the sacrificial interim layer, the Ms value has a 6607

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total amount of β-casein was decreased to only 5 fmol·μL−1. This result indicated that the detection limit of this method is at the fmol·μL−1 level. With its great selectivity in mind, we used human saliva to further examine the performance of yolk−shell MSP@ZrO2 for identifying the phosphoproteins in real complex samples. Proteins extracted from saliva were digested with trypsin and incubated with yolk−shell MSP@ZrO2 for enriching generated phosphopeptides. The eluted phosphopeptides were then subjected to Nano-LC-MS/MS analysis. A total of 33 unique phosphopeptides containing 49 phosphorylation sites mapped to 33 different phosphoproteins were identified (Table S1, Supporting Information). To the best of our knowledge, this is the best result for investigation of phosphoproteome of human saliva using only a one-step enrichment method hitherto, which is much better than the previous reported approach utilizing zinc oxide-coated iron oxide magnetic nanoparticles as the adsorbent.49 These results clearly demonstrate the high selectivity and effectiveness of our approach in enrichment of phosphopeptides from real biological samples. 3.3. Application of the Synthetic Strategy To Prepare Hybrid Yolk−Shell MSP@ZrO2−TiO2. Besides getting the desired yolk−shell MSP@ZrO2 microspheres containing one kind of metal oxide, our approach is also applicable to fabricate MSP@metal−oxides that are constructed with more than one constituent. When a mixture of multiple precursors, such as Zr(OBu)4 and Ti(OBu)4, with a same feeding amount were used at the same time, hybrid composite microspheres including both ZrO2 and TiO2 could be obtained (the product is marked as yolk−shell MSP@ZrO2−TiO2-1). As shown in the TEM image of the resulting product before calcination (the product is marked as MSP@PMAA@PMAA−Zr(OH)4−Ti(OH)4-1) (Figure 6a), well-defined core/shell/shell structure could be distinctly observed. The presence of the signals of the element Zr and Ti in the EDX spectrum (Figure 6b) is indicative of the existence of both Zr(OH)4 and Ti(OH)4 in the microspheres. Furthermore, the TEM image combined with

Figure 5. MALDI mass spectra of the tryptic digest mixture of β-casein and BSA (with a molar ratio of β-casein to BSA of 1:100): (a) direct analysis, (b) analysis after enrichment using yolk−shell MSP@ZrO2. (c) MALDI mass spectrum of a tryptic digests of β-casein (5 nM, 500 μL) after enrichment. “*” indicates phosphorylated peptides, “#” indicates their dephosphorylated counterparts, and “●” indicates their doubly charged phosphorylated peptides.

Figure 6. (a) Representative TEM image of MSP@ PMAA@PMAA−Zr(OH)4−Ti(OH)4 (scale bar is 50 nm); (b) EDX spectrum of MSP@ PMAA@PMAA−Zr(OH)4−Ti(OH)4; (c, d, e) EDX elemental maps of Fe, Zr, and Ti, respectively. 6608

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Figure 7. (a, b) Representative TEM image of yolk−shell MSP@ZrO2−TiO2-1 (scale bar is 100 nm for a and 50 nm for b); EDX spectra of (c) yolk−shell MSP@ZrO2−TiO2-1 and (d) yolk−shell MSP@ZrO2−TiO2-2.

of yolk−shell MSP@ZrO2 and the phosphate groups of phosphopeptides, the yolk−shell MSP@ZrO2 exhibited excellent performance in selective enrichment of phosphopeptides from both lab-made and real biological samples. In addition, this method is universal and can be used to produce hybrid yolk−shell MSP@ZrO2−TiO2. We also believe that the resulting yolk−shell MSP@metal−oxide microspheres could find great performance in some other applications, such as highperformance catalysis.

EDX elemental mapping (Figure 6c−e) clearly reveals the core/shell/shell structure with a MSP core, an interim PMAA layer, and an outer PMAA−Zr(OH)4−Ti(OH)4 shell, wherein Fe is distributed in the core while Zr and Ti are spread in the whole outer shell. In addition, the EDX elemental mapping of Zr and Ti also indicate that Zr(OH)4 and Ti(OH)4 are randomly distributed in the outer shell and their loading amount is nearly the same, which agrees well with the feeding amount of Zr(OBu)4 and Ti(OBu)4. During calcination, the adsorbed Zr(OH) 4 and Ti(OH) 4 will react with the neighboring counterparts to form a hybrid shell containing both ZrO2 and TiO2, while the intermediate PMAA layer will be eliminated at the same time. As seen in Figure 7a and 7b, the yolk−shell MSP@ZrO2−TiO2-1 possessed similar morphology as yolk−shell MSP@ZrO2. In addition, the EDX spectrum shown in Figure 7c revealed that the content ratio of Zr and Ti element in yolk−shell MSP@ZrO2−TiO2-1 remained the same as that in MSP@PMAA@PMAA−Zr(OH)4−Ti(OH)4-1. We also tried to regulate the composition of the metal oxide shell by adjusting the feeding ratio of Zr(OBu)4 to Ti(OBu)4 while keeping their total dosage constant. The result is that we can precisely control the ratio of the resulting ZrO2 to TiO2 in the shell by means of changing the dosage ratio of Zr(OBu)4 to Ti(OBu)4. For instance, when the dosage ratio of Zr(OBu)4 to Ti(OBu)4 was adjusted from 1:1 to 1:2 (the product is marked as yolk−shell MSP@ZrO2−TiO2-2), the resulting content ratio of Zr to Ti in yolk−shell MSP@ZrO2−TiO2 is also changed from approximately 1:1 to 1:2 (Figure 7c and 7d).

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ASSOCIATED CONTENT

S Supporting Information *

Overview of observed phosphopeptides derived from tryptic digests of human saliva after enrichment with yolk−shell MSP@ZrO2 microspheres. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions §

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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4. CONCLUSION In summary, we presented a new facile and repeatable synthetic route for preparation of hierarchical yolk−shell MSP@ZrO2 microspheres with well-defined core/void/shell structure, a highly crystalline ZrO2 shell, and high saturation magnetization. The cross-linked PMAA sacrificial layer plays a key role in successful preparation of the target nanomaterial. With the help of the strong interaction between highly crystalline ZrO2 shell

ACKNOWLEDGMENTS

This work was supported by the National Science and Technology Key Project of China (2012AA020204 and 2012CB910602), National Science Foundation of China (grant nos. 21025519 and 51073040), and Shanghai Projects (grants Eastern Scholar, 11XD1400800, 13520720200, 13JC1400500, and B109). 6609

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(20) Hong, Y. J.; Son, M. Y.; Kang, Y. C. One-Pot Facile Synthesis of Double-Shelled SnO2 Yolk-Shell-Structured Powders by Continuous Process as Anode Materials for Li-ion Batteries. Adv. Mater. 2013, 25, 2279−2283. (21) Seh, Z. W.; Li, W. Y.; Cha, J. J.; Zheng, G. Y.; Yang, Y.; McDowell, M. T.; Hsu, P. C.; Cui, Y. fSulphur-TiO2 Yolk-Shell Nanoarchitecture with Internal Void Space for Long-Cycle LithiumSulphur Batteries. Nat. Commun. 2013, 4, DOI: 10.1038/ ncomms2327. (22) Wu, X. J.; Xu, D. S. Formation of Yolk/SiO2 Shell Structures Using Surfactant Mixtures as Template. J. Am. Chem. Soc. 2009, 131, 2774−2775. (23) Wu, X. J.; Xu, D. S. Soft Template Synthesis of Yolk/Silica Shell particles. Adv. Mater. 2010, 22, 1516−1520. (24) Cheng, D. M.; Zhou, X. D.; Xia, H. B.; Chan, H. S. O. Novel Method for the Preparation of Polymeric Hollow Nanospheres Containing Silver Cores with Different Sizes. Chem. Mater. 2005, 17, 3578−3581. (25) Lou, X. W.; Yuan, C. L.; Archer, L. A. Double-Walled SnO2 Nano-Cocoons with Movable Magnetic Cores. Adv. Mater. 2007, 19, 3328−3332. (26) Li, H. X.; Bian, Z. F.; Zhu, J.; Zhang, D. Q.; Li, G. S.; Huo, Y. N.; Li, H.; Lu, Y. F. Mesoporous Titania Spheres with Tunable Chamber Stucture and Enhanced Photocatalytic Activity. J. Am. Chem. Soc. 2007, 129, 8406−8407. (27) Chen, H. M.; Liu, R. S.; Asakura, K.; Lee, J. F.; Jang, L. Y.; Hu, S. F. Fabrication of Nanorattles with Passive Shell. J. Phys. Chem. B 2006, 110, 19162−19167. (28) Vasquez, Y.; Sra, A. K.; Schaak, R. E. One-Pot Synthesis of Hollow Superparamagnetic CoPt Nanospheres. J. Am. Chem. Soc. 2005, 127, 12504−12505. (29) Yin, Y. D.; Rioux, R. M.; Erdonmez, C. K.; Hughes, S.; Somorjai, G. A.; Alivisatos, A. P. Formation of Hollow Nanocrystals through the Nanoscale Kirkendall Effect. Science 2004, 304, 711−714. (30) Kamata, K.; Lu, Y.; Xia, Y. N. Synthesis and Characterization of Monodispersed Core-Shell Spherical Colloids with Movable Cores. J. Am. Chem. Soc. 2003, 125, 2384−2385. (31) Chen, D.; Li, L. L.; Tang, F. Q.; Qi, S. O. Facile and Scalable Synthesis of Tailored Silica “Nanorattle” Structures. Adv. Mater. 2009, 21, 3804−3807. (32) Zhu, J. X.; Sun, T.; Hng, H. H.; Ma, J.; Boey, F. Y. C.; Lou, X. W.; Zhang, H.; Xue, C.; Chen, H. Y.; Yan, Q. Y. Fabrication of CoreShell Structure of M@C (M = Se, Au, Ag2Se) and Transformation to Yolk-Shell Structure by Electron Beam Irradiation or Vacuum Annealing. Chem. Mater. 2009, 21, 3848−3852. (33) Lou, X. W.; Yuan, C. L.; Archer, L. A. Shell-by-Shell Synthesis of Tin Oxide Hollow Colloids with Nanoarchitectured Walls: Cavity Size Tuning and Functionalization. Small 2007, 3, 261−265. (34) Li, G. L.; Kang, E. T.; Neoh, K. G.; Yang, X. L. Concentric Hollow Nanospheres of Mesoporous Silica Shell-Titania Core from Combined Inorganic and Polymer Syntheses. Langmuir 2009, 25, 4361−4364. (35) Ptacek, J.; Devgan, G.; Michaud, G.; Zhu, H.; Zhu, X. W.; Fasolo, J.; Guo, H.; Jona, G.; Breitkreutz, A.; Sopko, R.; McCartney, R. R.; Schmidt, M. C.; Rachidi, N.; Lee, S. J.; Mah, A. S.; Meng, L.; Stark, M. J. R.; Stern, D. F.; De Virgilio, C.; Tyers, M.; Andrews, B.; Gerstein, M.; Schweitzer, B.; Predki, P. F.; Snyder, M. Global Analysis of Protein Phosphorylation in Yeast. Nature 2005, 438, 679−684. (36) Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M. Global, in vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks. Cell 2006, 127, 635−648. (37) Huttlin, E. L.; Jedrychowski, M. P.; Elias, J. E.; Goswami, T.; Rad, R.; Beausoleil, S. A.; Villen, J.; Haas, W.; Sowa, M. E.; Gygi, S. P. A Tissue-Specific Atlas of Mouse Protein Phosphorylation and Expression. Cell 2010, 143, 1174−1189. (38) Swami, M. PROTEOMICS A Discovery Strategy for Novel Cancer Biomarkers. Nat. Rev. Cancer 2010, 10, 597−597.

REFERENCES

(1) Pellegrino, T.; Kudera, S.; Liedl, T.; Javier, A. M.; Manna, L.; Parak, W. J. On the Development of Colloidal Nanoparticles towards Multifunctional Structures and Their Possible Use for Biological Applications. Small 2005, 1, 48−63. (2) Kim, J.; Piao, Y.; Hyeon, T. Multifunctional Nanostructured Materials for Multimodal Imaging, and Simultaneous Imaging and Therapy. Chem. Soc. Rev. 2009, 38, 372−390. (3) Gao, J. H.; Gu, H. W.; Xu, B. Multifunctional Magnetic Nanoparticles: Design, Synthesis, and Biomedical Applications. Acc. Chem. Res. 2009, 42, 1097−1107. (4) Shi, D. L.; Bedford, N. M.; Cho, H. S. A Versatile Multicomponent Assembly via -cyclodextrin HostGuest Chemistry on Graphene for Biomedical Applications. Small 2011, 7, 2549−2567. (5) Dong, H. Q.; Li, Y. Y.; Yu, J. H.; Song, Y. Y.; Cai, X. J.; Liu, J. Q.; Zhang, J. M.; Ewing, R. C.; Shi, D. L. Engineered Multifunctional Nanocarriers for Cancer Diagnosis and Therapeutics. Small 2013, 9, 446−456. (6) 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. (7) Guo, J.; Yang, W. L.; Wang, C. C. Magnetic Colloidal Supraparticles: Design, Fabrication and Biomedical Applications. Adv. Mater. 2013, 25, 5196−5214. (8) Liu, J.; Qiao, S. Z.; Chen, J. S.; Lou, X. W.; Xing, X. R.; Lu, G. Q. Yolk/Shell Nanoparticles: New Platforms for Nanoreactors, Drug Delivery and Lithium-Ion Batteries. Chem. Commun. 2011, 47, 12578− 12591. (9) Hong, Y. J.; Son, M. Y.; Park, B. K.; Kang, Y. C. One-Pot Synthesis of Yolk-Shell Materials with Single, Binary, Ternary, Quaternary, and Quinary Systems. Small 2013, 9, 2224−2227. (10) Gao, J. H.; Liang, G. L.; Cheung, J. S.; Pan, Y.; Kuang, Y.; Zhao, F.; Zhang, B.; Zhang, X. X.; Wu, E. X.; Xu, B. Multifunctional YolkShell Nanoparticles: A Potential MRI Contrast and Anticancer Agent. J. Am. Chem. Soc. 2008, 130, 11828−11833. (11) Zhu, Y. F.; Ikoma, T.; Hanagata, N.; Kaskel, S. Rattle-Type Fe3O4@SiO2 Hollow Mesoporous Spheres as Carriers for Drug Delivery. Small 2010, 6, 471−478. (12) Lee, J.; Park, J. C.; Song, H. A Nanoreactor Framework of a Au@SiO2 Yolk/Shell Structure for Catalytic Reduction of p-Nitrophenol. Adv. Mater. 2008, 20, 1523−1528. (13) Liu, J.; Qiao, S. Z.; Hartono, S. B.; Lu, G. Q. Monodisperse Yolk-Shell Nanoparticles with a Hierarchical Porous Structure for Delivery Vehicles and Nanoreactors. Angew. Chem., Int. Ed. 2010, 49, 4981−4985. (14) Li, W.; Deng, Y. H.; Wu, Z. X.; Qian, X. F.; Yang, J. P.; Wang, Y.; Gu, D.; Zhang, F.; Tu, B.; Zhao, D. Y. Hydrothermal Etching Assisted Crystallization: A Facile Route to Functional Yolk-Shell Titanate Microspheres with Ultrathin Nanosheets-Assembled Double Shells. J. Am. Chem. Soc. 2011, 133, 15830−15833. (15) Yang, Y.; Liu, J.; Li, X. B.; Liu, X.; Yang, Q. H. OrganosilaneAssisted Transformation from Core-Shell to Yolk-Shell Nanocomposites. Chem. Mater. 2011, 23, 3676−3684. (16) Liu, J.; Yang, H. Q.; Kleitz, F.; Chen, Z. G.; Yang, T. Y.; Strounina, E.; Lu, G. Q.; Qiao, S. Z. Yolk-Shell Hybrid Materials with a Periodic Mesoporous Organosilica Shell: Ideal Nanoreactors for Selective Alcohol Oxidation. Adv. Funct. Mater. 2012, 22, 591−599. (17) Yang, Y.; Liu, X.; Li, X. B.; Zhao, J.; Bai, S. Y.; Liu, J.; Yang, Q. H. A Yolk-Shell Nanoreactor with a Basic Core and an Acidic Shell for Cascade Reactions. Angew. Chem., Int. Ed. 2012, 51, 9164−9168. (18) Kuo, C. H.; Tang, Y.; Chou, L. Y.; Sneed, B. T.; Brodsky, C. N.; Zhao, Z. P.; Tsung, C. K. Yolk-Shell Nanocrystal@ZIF-8 Nanostructures for Gas-Phase Heterogeneous Catalysis with Selectivity Control. J. Am. Chem. Soc. 2012, 134, 14345−14348. (19) Liu, N.; Wu, H.; McDowell, M. T.; Yao, Y.; Wang, C. M.; Cui, Y. A Yolk-Shell Design for Stabilized and Scalable Li-Ion Battery Alloy Anodes. Nano Lett. 2012, 12, 3315−3321. 6610

dx.doi.org/10.1021/la501381v | Langmuir 2014, 30, 6602−6611

Langmuir

Article

(39) Ashman, K.; Villar, E. L. Phosphoproteomics and Cancer Research. Clin. Transl. Oncol. 2009, 11, 356−362. (40) Hu, L. H.; Zhou, H. J.; Li, Y. H.; Sun, S. T.; Guo, L. H.; Ye, M. L.; Tian, X. F.; Gu, J. R.; Yang, S. L.; Zou, H. F. Profiling of Endogenous Serum Phosphorylated Peptides by Titanium (IV) Immobilized Mesoporous Silica Particles Enrichment and MALDITOFMS Detection. Anal. Chem. 2009, 81, 94−104. (41) Ma, W. F.; Zhang, Y.; Li, L. L.; You, L. J.; Zhang, P.; Zhang, Y. T.; Li, J. M.; Yu, M.; Guo, J.; Lu, H. J.; Wang, C. C. Tailor-Made Magnetic Fe3O4@mTiO2 Microspheres with a Tunable Mesoporous Anatase Shell for Highly Selective and Effective Enrichment of Phosphopeptides. ACS Nano 2012, 6, 3179−3188. (42) Li, Y.; Leng, T. H.; Lin, H. Q.; Deng, C. H.; Xu, X. Q.; Yao, N.; Yang, P. Y.; Zhang, X. M. Preparation of Fe3O4@ZrO2 Core-Shell Microspheres as Affinity Probes for Selective Enrichment and Direct Determination of Phosphopeptides Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. J. Proteome Res. 2007, 6, 4498−4510. (43) Zhang, Y. T.; Li, L. L.; Ma, W. F.; Zhang, Y.; Yu, M.; Guo, J.; Lu, H. J.; Wang, C. C. Two-in-One Strategy for Effective Enrichment of Phosphopeptides Using Magnetic Mesoporous gamma-Fe2O3 Nanocrystal Clusters. ACS Appl. Mater. Interfaces 2013, 5, 614−621. (44) Larsen, M. R.; Jensen, S. S.; Jakobsen, L. A.; Heegaard, N. H. H. Exploring the Sialiome Using Titanium Dioxide Chromatography and Mass Spectrometry. Mol. Cell. Proteomics 2007, 6, 1778−1787. (45) Ma, W. F.; Xu, S.; Li, J. M.; Guo, J.; Lin, Y.; Wang, C. C. Hydrophilic Dual-Responsive Magnetite/PMAA Core/Shell Microspheres with High Magnetic Susceptibility and pH Sensitivity via Distillation-Precipitation Polymerization. J. Polym. Sci., Part A: Polym. Chem. 2011, 49, 2725−2733. (46) Jin, S.; Pan, Y. J.; Wang, C. C. Reflux Precipitation Polymerization: A New Technology for Preparation of Monodisperse Polymer Nanohydrogels. Acta Chim. Sin. 2013, 71, 1500−1504. (47) 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. (48) Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. Detection and Sequencing of Phosphopeptides Affinity Bound to Immobilized Metal Ion Beads by Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2000, 11, 273−282. (49) Chen, W. Y.; Chen, Y. C. Functional Fe3O4@ZnO Magnetic Nanoparticle-Assisted Enrichment and Enzymatic Digestion of Phosphoproteins from Saliva. Anal. Bioanal. Chem. 2010, 398, 2049−2057.

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