Soft-template synthesis of hydrophilic metallic zirconia nanoparticle-incorporated ordered mesoporous carbon composite and its application in phosphopeptide enrichment

Abstract

Novel nanostructure metallic zirconia incorporated ordered mesoporous carbon (OMC) composites were synthesized via a multi-component co-assembly strategy associated with a direct carbonization process from resol, ZrO(NO₃)₂·xH₂O, and triblock copolymer F127. Herein, the novel metallic zirconia incorporated OMC as a Metal Oxide Affinity Chromatography (MOAC) material had large mesopores (5.6 nm), a high surface area (387 m² g⁻¹), a large pore volume (0.35 cm³ g⁻¹) and excellent hydrophilicity for the selective enrichment of phosphopeptides. The surface construction and chemical composition of the material were characterized by SEM, TEM, XPS, EDS, BET, and XRD. Experimental conditions for trapping phosphopeptides were optimized using β-casein as the standard protein. Under the optimal conditions combined with the merits of high surface area of material and special combination with phosphopeptides, the zirconia/OMC composites for enrichment of the phosphopeptides showed a high selectivity (phosphopeptides/non-phosphopeptides at a molar ratio of 1 : 300) and lower detection sensitivity (1.5 fmol). Moreover, the as-prepared MOAC zirconia/OMC composites were successfully applied to the detection and identification of low-abundance phosphopeptides from non-fat milk.

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Introduction

Phosphorylation is one of the most important of post-translational modifications.¹ It is recognized as playing a key role in living such as the cell cycle, cell growth, cell differentiation and metabolism.² However, determination of phosphorylation sites with mass spectrometry is still under research because of the low abundance of phosphoproteins and interference by high-abundance proteins in the complex sample. To date, there are a lot of strategies have been developed for the enrichment of phosphopeptides such as immobilized metal ion affinity chromatography (IMAC),^3–6 antibodies,⁷ chemical modification,^8,9 strong cation exchange (SCX),¹⁰ strong anion exchange (SAX)¹¹ and metal oxide affinity chromatography (MOAC).^12–15 Among them, MOAC is one of the most commonly used techniques.¹⁶ In the MOAC method, phosphopeptides can be firmly adsorbed onto the surface of metal oxides, such as TiO₂,¹⁷ ZrO₂,¹⁸ Fe₂O₃,¹⁹ Ta₂O₅ ²⁰ in an acidic environment via bridging bidentate interaction between the phosphate groups and the surface of the metal oxides while being easily desorbed from the surface in alkaline conditions.

For MOAC, there are many strategies for metal oxides combined with matrices such as magnetic Fe₃O₄, graphene oxide and yolk–shell structural magnetic nanocomposites in phosphopeptide enrichment. For example, Ma²¹ et al. has synthesized Fe₃O₄@mTiO₂ by a direct and simple coating method using the matrix of magnetic Fe₃O₄. Wang²² et al. has reported a simple approach to prepare Fe₃O₄@TiO₂–ZrO₂ for phosphopeptide enrichment. Huang²³ et al. reported a graphene oxide induced growth of fusiform zirconia nanostructures for capturing phosphopeptides. Wan²⁴ et al. has synthesized magnetic yolk–shell Fe₃O₄@mTiO₂@mSiO₂ nanocomposites for selective enrichment of endogenous phosphopeptides.

Ordered mesoporous carbon (OMC) as a type of carbon based material, has attracted considerable attention due to its advantages of good electrical conductivity, thermal stability, continuous channels, uniform pore size (2–50 nm), regularly aligned architecture, high specific surface area (up to 2500 m² g⁻¹), and large pore volume for supporting metal nanoparticles.²⁵ The OMC and metal-oxide-containing OMC materials have been widely used as adsorbents, catalysts, drug delivery carriers and electrodes. Yang²⁶ et al. has reported a nano copper oxide-incorporated OMC composite as an adsorbent for the selective separation of hemoglobin. Yu²⁷ et al. has synthesized titanium carbide nanoparticle-containing mesoporous carbon.

Herein, we report novel hydrophilic nanostructure metallic zirconia incorporated ordered mesoporous carbon composites. The synthesis is based on a multi-component co-assembly process which is accomplished by slow evaporation of an ethanol solution containing soluble resol as a carbon source, ZrO(NO₃)₂·xH₂O as a metallic precursor, acetylacetone as a chelating agent, and triblock copolymer F127 as a soft template. The obtained nanocomposites have a 2-D hexagonally arranged pore structure uniform pore size, high surface area, moderate pore volume and uniform and highly dispersed metallic oxides. Because of the metallic zirconia highly dispersed ordered mesoporous carbon and excellent hydrophilicity, the material is suitable for enrichment of phosphopeptides.

Experimental section

Materials and reagents

Poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) triblock copolymer Pluronic F127 (PEO₁₀₆PPO₇₀PEO₁₀₆, M_w = 12 600) was purchased from Sigma-Aldrich (USA). Phenol, ethanol, acetylacetone, ammonium hydroxide, hydrochloric acid, sodium hydroxide, ammonium hydrogen carbonate, formalin solution (37 wt%) were analytical reagent grade and obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). ZrO(NO₃)₂·xH₂O was from Aladdin Chemical Reagent Co., Ltd (Shanghai, China). β-Casein (from bovine milk), BSA, trypsin (TPCK treated), dithiothreitol (DTT), iodoacetamide (IAA), urea, trifluoroacetic acid (TFA), acetonitrile (ACN) and 2,5-dihydroxyl benzoic acid (DHB) were purchased from Sigma-Aldrich (USA). These reagents were at least of analytical-reagent grade and used as received without further treatment. Non-fat milk was obtained from a local supermarket.

Characterization

The metallic zirconia incorporated ordered mesoporous carbon composite structure and the surface morphology of the microspheres were studied by ultrahigh resolution field emission scanning electron microscopy (UHRFESEM, NOVA Nano SEM450, FEI, USA) and ultrahigh resolution transmission electron microscopy (HR-TEM, JEM-2100, JEOL, Japan). The crystalline structure of the material was characterized by D/MAX 2550 VB/PC advance X-ray powder diffraction (XRD, RIGAKU, Japan) with a Cu Kα source. The 2θ angles probed were from 10° to 80° at a rate of 5° min⁻¹. The X-ray photoelectron spectra were obtained using an ESCALAB 250Xi X-ray photoelectron spectrometer (XPS). The material element contents were determined by an energy dispersive spectrometer (EDS, Falcon, EDAX, USA). Nitrogen sorption/desorption isotherm was measured at 77 K with a Micromeritics Instrument Corporation TriStar II 3020 (USA). Before the sorption/desorption measurement, the samples were degassed in a vacuum at 300 °C for 10 h. The Brunauer–Emmett–Teller (BET) method was used to calculate the specific surface area according to the adsorption data in a relative pressure from 0.06 to 0.30. The pore size distribution (PSD) was calculated based on the adsorption branch by using the Barrett–Joyner–Halenda (BJH) model. The quantification of Zr in the OMC composite was performed using inductively coupled plasma spectrometry with a Radial ICP-OES instrument (ICP-OES, Agilent 725, USA).

Mass spectrometry analysis

All MALDI-TOF MS experiments were performed in the reflector positive mode in an AB Sciex 4800 plus MALDI-TOF MS/MS mass spectrometer (AB Sciex, CA) with a pulsed Nd/YAG laser at 355 nm. The DHB matrix was dissolved in ACN–H₂O–H₃PO₄ (70 : 29 : 1, (v/v/v), 25 mg). A 0.5 μL aliquot of the eluate and 0.5 μL of the DHB matrix were sequentially dropped onto the MALDI plate for MS analysis.

Preparation of the zirconia/OMC composite

Soluble resol precursors were prepared by using phenol and formaldehyde via a base-catalyzed procedure according to the procedure previously reported.²⁸ The synthesis procedure in brief was the following: 10 g phenol was heated to melt at 42 °C, then 2.13 g NaOH solution (20 wt%) was added slowly under stirring. After that, 17.7 g formalin solution (37 wt%) was added dropwise. The obtained mixture was heated at 75 °C with stirring for 60 min. After the mixture was cooled down to room temperature, it was adjusted to pH 6 with hydrochloric acid solution. The mixture solution was dried by vacuum distillation at 50 °C and centrifuged for removal of the produced NaCl. Then, the obtained resol precursor was redissolved in ethanol (20 wt%) for use.

The metallic zirconia incorporated ordered mesoporous carbon composites were synthesized through the chelate-assisted multi-component co-assembly strategy.²⁸ The preparation was accomplished by gentle evaporation of an ethanol solution containing Pluronic F127, resol, acetylacetone, and ZrO(NO₃)₂·xH₂O. In general, 0.5 g Pluronic F127 was dissolved in 7.0 g absolute ethanol. Then, 2.5 g resol precursor solution was added and the mixture was stirred for 10 min. 0.234 g ZrO(NO₃)₂·xH₂O was dissolved in 1.75 g ethanol. After that, 0.025 g acetylacetone solution was sequentially added dropwise into the above mixture. After stirring for 30 min, the mixture was cast into a Petri dish, followed by evaporation of ethanol at room temperature for 12 h. Then the generated sticky film was heated at 100 °C for 24 h. The resulting film was scraped off and calcination followed in a tube furnace at 600 °C for 3 h at a temperature ramp of 1 °C min⁻¹ under a N₂ atmosphere. During the carbonization of the resol precursor in the calcination process, zirconia nanocrystallites were generated in situ.

Tryptic digestion of standard protein

1 mg β-casein was dissolved in NH₄HCO₃ solution (1 mL, 50 mmol L⁻¹) and denatured by boiling for 10 min. Then, protein solution was incubated with trypsin (an enzyme/protein ratio of 1 : 40, w/w) for 16 h at 37 °C. The tryptic peptide mixtures were stored at −20 °C until further use. The 2 mg BSA was denatured in urea (1 mL, 8 mol L⁻¹) and NH₄HCO₃ solution (1 mL, 50 mmol L⁻¹), followed by addition of DTT (20 μL, 1 mol L⁻¹) and incubating at 60 °C for 1 h. Subsequently, 7.4 mg IAA was added and incubated at room temperature in the dark for 40 min. The mixture was diluted ten-fold with NH₄HCO₃ solution (50 mmol L⁻¹). Then, the protein was subjected to enzymolysis with trypsin using the same procedure as for β-casein. The tryptic peptide solution was desalted by C18 SPE and freeze-dried until further use.

Tryptic digestion of proteins extracted from non-fat milk

30 μL of non-fat milk was added into NH₄HCO₃ solution (1 mL, 25 mmol L⁻¹) and centrifuged at 16 000 rpm for 15 min. The supernatant was collected and denatured by boiling for 10 min. The supernatant was digested with trypsin (40 μg) at 37 °C for 16 h.

Enrichment of phosphopeptides

200 μg of zirconia/OMC composites were added into the loading buffer (ACN–H₂O–TFA, 90 : 5 : 5, (v/v/v), 400 μL) containing β-casein tryptic digest, BSA tryptic digest and proteins extracted from non-fat milk. The mixture was gently incubated at room temperature for 30 min. After removing the supernatant, the zirconia/OMC composites were washed three times with the loading buffer. The captured phosphopeptides were eluted by ammonium hydroxide (20 μL, 10 wt%) by vigorous shaking for 15 min. The eluate was directly analyzed by MALDI-TOF MS.

Results and discussion

Preparation and characterization of zirconia/OMC composite

The preparation protocol of metallic zirconia embedded in the ordered mesoporous carbon is illustrated in Scheme 1. In this protocol, firstly, a soluble phenolic resin precursor (resol) was synthesized by a base-catalyzed procedure. Then, the ordered mesoporous carbon with incorporated nanoparticles was synthesized by a soft-templating approach based on a chelate-assisted solvent evaporation induced co-assembly strategy.

Scheme 1 Synthetic procedure of metallic zirconia incorporated ordered mesoporous carbon.

In the co-assembly system, a soluble low-molecular weight polymer of resol was used as the carbon source with an amphiphilic triblock copolymer Pluronic F127 as the template, ZrO(NO₃)₂·xH₂O as the metallic source and acetylacetone as the coupling reagent. Finally, the decomposition film was pyrolyzed at 600 °C under Na ₂ atmosphere to remove template F127 and form the highly ordered and aligned mesopores, and meanwhile, the resol framework was carbonized into mesoporous carbon accompanied by in situ growth of zirconia nanoparticles.

The HR-TEM image (Fig. 1a) and selected area electron diffraction of TEM (Fig. S1 ESI†) show the zirconia/OMC composites have a 2-D hexagonally arranged porous and ordered structure. The domain of the pore size is estimated to be 4.0–6.0 nm. The metallic zirconia nanoparticles are uniformly dispersed within the ordered pore walls in large domains without aggregation (inset Fig. 1a). The Energy Dispersive X-ray Spectroscopy (EDS) by TEM (Fig. 1b), indicated that the Zr element existed in the OMC. The quantification of Zr element (3.5 wt%) was determined by ICP-OES, indicating that the uniformly dispersed nanoparticles were metallic zirconia and embedded in the OMC.

Fig. 1 HR-TEM (a, left) and EDS by TEM (b, right) of metallic zirconia incorporated ordered mesoporous carbon.

XRD patterns of the zirconia/OMC composites are shown in Fig. 2. Between the wide-angle range from 10° to 80°, the diffraction peaks (2θ) (Fig. 2a) at 30.12°, 51.34° and 60.01° might be assigned to the reflections of the tetragonal phase ZrO₂ (JCPDS card 50-1089) and the reflections of the orthorhombic phase ZrO₂ (JCPDS card 79-1796). The wide-angle XRD pattern clearly indicates that the in situ grown metal nanoparticles are well crystallized ZrO₂. The small-angle XRD pattern has a typical mesoporous structural diffraction peak from Fig. 2b, which indicates the carbon has a 2-D hexagonally arranged mesoporous structure.

Fig. 2 XRD patterns of metallic zirconia incorporated ordered mesoporous carbon. (a) Wide-angle and (b) small-angle.

XPS spectra have been widely used to identify the existence of a particular element on the surface of a material. The XPS spectra of the zirconia/OMC composites are shown in Fig. 3. The XPS spectra confirmed the existence of C, O, Zr, Na on the surface of the zirconia/OMC composites. In Fig. 3a, the peaks of C1s at around 284.6 eV, O1s at around 532.6 eV, Zr3d at around 180.07 eV, and Na1s at around 1071.00 eV were observed. This demonstrated that the zirconia nanocrystallites were in situ generated on the mesoporous carbon. The residual sodium salt appeared on the composites. By the XPS of Zr3d, the peaks at about 180.17 eV and 182.48 eV are shown in Fig. 3b, which are in agreement with the ZrO₂ standard data.²⁹ So, the XPS characterization revealed that nano ZrO₂ was incorporated into ordered mesoporous carbon.

Fig. 3 XPS of metallic zirconia incorporated ordered mesoporous carbon (a) and the XPS of Zr_3d (b).

N₂ sorption/desorption curves in Fig. 4 illustrate typical Langmuir-IV sorption–desorption isotherms with H₂ hysteresis loops and large uptake at a low relative pressure for zirconia/OMC composites. The hysteresis loop (capillary condensation) in a range of P/P₀ = 0.3–0.8 is attributed to the existence of irregular mesopores formed on the zirconia/OMC composite surface. The Langmuir-IV sorption–desorption isotherms curves indicate the presence of uniform mesopores which are attributed to the removal of template F127 and calcination of the resol framework at a high temperature. The BET surface area and pore volume of the obtained zirconia/OMC composites are calculated to be 387 m² g⁻¹ and 0.35 cm³ g⁻¹. The pore size distribution curves derived from the adsorption branches using the BJH model also reveal the uniform mesopores (Fig. 4b). The mean size of the mesopore is located in the range of 3.8–7.8 nm, and the most probable aperture is ca. 5.6 nm.

Fig. 4 N₂ sorption/desorption isotherms (a, left) and pore size distribution curve (b, right) of metallic zirconia incorporated ordered mesoporous carbon.

As is well known, most ordered mesoporous carbon materials are hydrophobic, but the zirconia/OMC composites have excellent hydrophilicity. The material can be uniformly dispersed in water (Fig. 5a) without precipitation after ten days. However, when the composites were dispersed in oil petroleum ether (Fig. 5b), the particles aggregated together and precipitated at the bottom of the centrifuge tube after 30 seconds. This indicated that the zirconia/OMC composites had excellent hydrophilicity and would be beneficial to the enrichment of phosphopeptides in soluble biological samples. The OMC without zirconia showed hydrophilicity too, which might be from oxidation of the carbon material. But better hydrophilicity was observed after incorporation of the metallic zirconia nanoparticles. Zirconia is known to have amphoteric properties and has excellent hydrophilicity, which can react either as a Lewis acid or Lewis base depending on the pH of the reaction solution. Then the excellent hydrophilicity of zirconia/OMC nanocomposites may arise from the joint effect of oxidation of ordered mesoporous carbon and hydrophilic metallic zirconia.

Fig. 5 The hydrophilicity experience of the metallic zirconia incorporated ordered mesoporous carbon. (a) Dispersed in water and (b) dispersed in oil petroleum ether.

Application to phosphopeptide enrichment

To demonstrate the practicability of the zirconia/OMC composites as a MOAC stationary phase for the enrichment of phosphopeptides, the enrichment of the standard phosphoprotein (bovine β-casein) tryptic digest was studied. β-Casein tryptic digest was incubated with zirconia/OMC composites in loading buffer, then after enrichment and elution, the captured phosphopeptides were deposited on the MALDI target for MALDI-TOF MS analysis. As shown in Fig. 6a, for the direct analysis of β-casein tryptic digest, because of the low-concentration of phosphopeptides and severe signal suppression by abundant non-phosphopeptides, only one phosphopeptide with a weak MS signal intensity and low signal-to noise (S/N) ratio was detected. However, after enrichment by zirconia/OMC composites (Fig. 6b), the three expected phosphopeptides (β₁, β₂, and β₃) could be detected clearly with strong MS signal intensities and S/N ratio, along with dephosphopeptides, which were likely formed during the MALDI ionization process. Meanwhile, another dephosphopeptide β₄ (m/z 2353.39) was detected. Phosphopeptides α_S2 (m/z 1252.57) which originated from α-casein were also clearly detected with strong MS signal intensities and S/N ratio. The detailed information of the captured phosphopeptides from β-casein is displayed in Table S2 ESI.† By contrast, the OMC without incorporated metallic zirconia (Fig. 6c) was used to enrich the β-casein tryptic digest. There was no peak representative of the existence of phosphopeptide.

Fig. 6 MADIL-TOF mass spectra of the tryptic digests of β-casein. (a) Direct analysis (0.5 pmol) and (b) after enrichment by metallic zirconia incorporated ordered mesoporous carbon (1.0 pmol) and (c) ordered mesoporous carbon without metallic zirconia (0.5 pmol). ★ indicates phosphopeptide, # indicates dephosphopeptide, the β indicates phosphopeptide was from β-casein and α★ indicates phosphopeptide was from α-casein.

To evaluate the high selectivity of the IMAC materials for the enrichment of phosphopeptides, a mixture of β-casein and BSA tryptic digest was used as the test sample. As shown in Fig. 7, different molar ratios of β-casein and BSA (1 : 100 (a), 1 : 200 (b) and 1 : 300 (c)) were used to evaluate the selectivity of the zirconia/OMC composites. From Fig. 7c, when the molar ratio of β-casein and BSA was decreased to 1 : 300, the two target phosphopeptides could still be distinctly identified with a clean background. These results indicated that the zirconia/OMC composites have high selectivity in a complex peptide mixture.

Fig. 7 MALDI-TOF mass spectra of the tryptic digest mixtures of β-casein (0.5 pmol) and BSA after enrichment by metallic zirconia incorporated ordered mesoporous carbon at molar ratios of (a) 1 : 100, (b) 1 : 200, and (c) 1 : 300. ★ indicates phosphopeptide.

Because of the low abundance of phosphopeptides and complex matrix in the biological sample, the ability of enriching and detecting phosphopeptides from a highly diluted solution is a key parameter to evaluate the enrichment performance of the MOAC materials. So, different amounts of β-casein tryptic digest were used to evaluate the ability of zirconia/OMC composites. As shown in Fig. 8a, the peak m/z of 2016 was clearly detected in 150 fmol of β-casein tryptic digest with the S/N ratio of 157. When the concentration was decreased to 15 fmol (Fig. 7b), it still had a high S/N ratio of 134. Even though the total amount of β-casein tryptic digest was as low as 1.5 fmol, it also could detect one phosphopeptide with a S/N ratio of 13. The resulting detection sensitivity was higher than those of many previously reported MOAC nanomaterials such as Fe₃O₄@mTiO₂ (10 fmol),²¹ hollow Al₂O₃ spheres (5 fmol),³⁰ and mesoporous γ-Fe₂O₃ (50 fmol).¹⁹ The lower detection limit may be attributed to the excellent hydrophilicity and high loaded amount of metallic zirconia. The detection limit experience indicated that the zirconia/OMC composites have a high detection sensitivity for phosphopeptide.

Fig. 8 MALDI-TOF mass spectra of the tryptic digests of β-casein after enrichment by metallic zirconia incorporated ordered mesoporous carbon. (a) 150 fmol (0.5 μL), (b) 15 fmol (0.5 μL) and (c) 1.5 fmol (0.5 μL). ★ indicates phosphopeptide.

Application to enrichment of phosphopeptides from non-fat milk

To examine the effectiveness of the zirconia/OMC composites in selective enrichment of low abundance phosphopeptides from real biological samples, non-fat milk was used as a real sample. The direct analysis results of the digested non-fat milk by MALDI-TOF MS are shown in Fig. 9a, where only several weak MS signal intensities of phosphopeptides can be detected due to the interference of abundant non-phosphopeptides. However, after enrichment by zirconia/OMC composites (Fig. 9b), lots of peaks of phosphopeptides are clearly observed with a clean background. Through searching and contrasting the literature,^6,31 fifteen MS peaks of phosphopeptides were identified including seven mono-phosphopeptides and eight multi-phosphopeptides. The detailed information of the phosphopeptides from the tryptic digest of proteins extracted from non-fat milk is given in Table S3 ESI.† The results indicated that zirconia/OMC composites had the ability of highly selective capturing of phosphopeptides from a naturally complex biological sample. Compared with other zirconia-decorated nanomaterials in the literature (Table S4, ESI†), we achieved a relatively lower detection limit, higher selectivity and more identified phosphopeptides.

Fig. 9 MALDI-TOF mass spectra of the tryptic digests of non-fat milk. (a) Direct analysis and (b) after enrichment by metallic zirconia incorporated ordered mesoporous carbon. ★ indicates phosphopeptide.

The reproducibility and stability of the metallic zirconia incorporated ordered mesoporous carbon for enrichment of phosphopeptides were investigated by the standard phosphoprotein (bovine β-casein) tryptic digest. After the zirconia/OMC was stored in water for about six months, the phosphopeptides from the β-casein tryptic digest can still be selectively captured (four consecutive enrichments). The detailed experimental results are shown in ESI (Fig. S4†). The results indicated great stability and reproducibility of the zirconia/OMC composites.

Conclusions

In this work, novel hydrophilic nanostructure metallic zirconia incorporated ordered mesoporous carbon composites were synthesized via a soft template of multi-component co-assembly strategy. The zirconia/OMC composites had many advantages such as large mesopores (5.6 nm), high surface area (387 m² g⁻¹), large pore volume (0.35 cm³ g⁻¹), high loading content of metallic zirconia (3.5 wt%) and high hydrophilicity. The hydrophobic OMC was changed to be hydrophilic though. Because of the higher capacity of metallic zirconia and the excellent hydrophilicity, it improved the selectivity (1 : 300) and sensitivity (1.5 fmol) for enrichment of phosphopeptides. In the selective enrichment of phosphopeptide from non-fat milk, the metallic zirconia incorporated ordered mesoporous carbon displays excellent practicability in identifying low abundance phosphopeptides from complex biological samples.

Acknowledgements

This work was financially supported by the National 973 Program (2011CB910403), the National Key Scientific Instrument and Equipment Development Project (2012YQ120044), the National Natural Science Foundation of China (21475044) and foundation of Shanghai Research Institute of Criminal Science and Technology.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra00326e

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