Facile preparation of graphene/Fe₃O₄/TiO₂ multifunctional composite for highly selective and sensitive enrichment of phosphopeptides

Abstract

A novel multifunctional graphene/Fe₃O₄/TiO₂ composite with excellent hydrophilicity and biological compatibility was synthesized and applied to the fast, highly selective and sensitive enrichment of phosphopeptides from biosamples.

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Protein phosphorylation is one of the most significant post-translational modifications (PTMs) in nature, playing a crucial role in eukaryotic cells, involved in many regulatory functions, such as cell growth, differentiation, division, signal transduction and metabolism.¹ Therefore, a comprehensive analysis of protein phosphorylation via the identification of phosphorylation sites is of keen interest in the field of proteomics.² In recent years, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray-ionization mass spectrometry (ESI-MS),³ have been widely employed to study PTMs owing to their high sensitivity and high-throughput. However, it is still a great challenge to detect phosphopeptides due to their low abundance and low ionization efficiency.⁴ Therefore, the selective enrichment of phosphoproteins or phosphopeptides from complex biosamples is an essential step prior to MS analysis.

To date, immobilized metal ion affinity chromatography (IMAC) has been widely applied to enrich the phosphopeptides. When loaded on IMAC, the chelating metal ions (Fe³⁺, Ga³⁺, Zr⁴⁺, Ti⁴⁺) bind specifically to the phosphopeptides from complex mixtures.⁵ Nevertheless, the limitation in IMAC-based enrichment is the high level of unspecific binding of acidic peptides.⁶ Metal oxide affinity chromatography (MOAC) has emerged to be an attractive alternative to IMAC.⁷ Although TiO₂-based MOAC materials have been the most commonly used for the enrichment of phosphopeptides, owing to its high chemical stability over a wide pH range, selectivity and recovery, and relatively high salt tolerance,⁸ the aggregation of TiO₂ particles limits the available surface area of adsorbent for the efficient enrichment.

Graphene, an sp²-bonded carbon sheet with the thickness of a single atom, has attracted a great deal of attention in recent years due to its unique mechanical, thermal, optical and electrical properties.⁹ With the inherent superiorities, graphene has been successfully utilized as a good adsorbent in sample pretreatment based on its ultrahigh specific surface area, high loading capacity, large delocalized π-electron system and hydrophobic interaction.¹⁰ However, its poor hydrophilicity and difficulty in the surface functionalization often limit its more extensive bioapplication. Recently, Ti⁴⁺ or TiO₂ immobilized graphene can be used as the adsorbent for the selective extraction of phosphopeptides from complex peptides mixtures.¹¹ The direct use of graphene as an adsorbent needs a complicated process to isolate the graphene and targets from the samples. Decorating magnetic iron oxide nanoparticles onto graphene in a composite will facilitate to impart the desirable magnetic property into graphene in a variety of application fields, such as bioseparation, medical diagnosis, magnetically targeted drug delivery, magnetic energy storage and catalysis.¹²

Herein, we designed a facile synthetic strategy of graphene/Fe₃O₄/TiO₂ multifunctional composite material for the enrichment of phosphopeptides (Scheme 1). The graphene/Fe₃O₄/TiO₂ multifunctional composite exhibited several attractive advantages. Firstly, the large specific surface area of graphene offers higher capacity for loading the TiO₂ nanoparticles and thus possesses the high available surface area for the efficient enrichment of phosphopeptides. Second, the hydrophilic polydopamine layer on the magnetic graphene exhibits excellent environmental stability, good biocompatibilty and good water dispersibility,¹³ and also decreases the non-specific adsorption of non-phosphopeptides. Finally, the highly loaded Fe₃O₄ nanoparticles accelerate the isolation. The resulting graphene/Fe₃O₄/TiO₂ composite can selectively capture, fast magnetically isolate and sequentially determine of the low-abundance phosphopeptides from the complex biosamples.

Scheme 1 Schematic illustration of the synthesis and enrichment of graphene/Fe₃O₄/TiO₂ multifunctional composite for phosphopeptides.

The synthetic strategy of graphene/Fe₃O₄/TiO₂ magnetic multifunctional composite and the procedure for the selective enrichment of phosphopeptides are illustrated in Scheme 1. The graphene/Fe₃O₄ composite was firstly prepared by a modified solvothermal method. The surface modified graphene oxide (GO) highly loaded with Fe₃O₄ nanoparticles is hydrothermally produced from the reduction reaction between FeCl₃ and ethylene glycol in the presence of GO. Then, the polydopamine (PDA)-capped graphene/Fe₃O₄ was prepared via the self-polymerization of dopamine hydrochloride. Dopamine, commonly known as a neuroendocrine transmitter and a unique molecule mimicking the adhesive proteins, exhibits excellent affinity for most organic and inorganic surfaces such as metal, metal oxide, and polymer surfaces and has been found to be able to polymerize into a unique hydrophilic PDA coating on a variety of substrates at a weak alkaline pH.¹⁴ Moreover, as a good reducing agent, dopamine has recently been used to fabricate nanocomposites via directly reacting with HAuCl₄.¹⁵ Finally, graphene/Fe₃O₄/TiO₂ multifunctional composites were obtained by the hydrolysis of Ti⁴⁺ immobilized on the PDA (see Experimental details in ESI†).

The TEM images of graphene/Fe₃O₄ and graphene/Fe₃O₄/TiO₂ composites are shown in Fig. 1. Obviously, the two-dimensional graphene sheets are well decorated by a large quantity of uniform spherical and evenly distributed Fe₃O₄ particles (with average size of about 150 nm). The TEM image of graphene/Fe₃O₄/TiO₂ (Fig. 1b) clearly reveals that the TiO₂ nanoparticles with average diameter of 2 nm highly loaded on the surface of PDA–graphene/Fe₃O₄. It is clear that the plane structure of graphene/Fe₃O₄ was well retained due to the good chemical and physical stability of graphene, and the functional layer of PDA was successfully coated on graphene and Fe₃O₄.

Fig. 1 TEM images of graphene/Fe₃O₄ (a) and graphene/Fe₃O₄/TiO₂ (b) magnetic composite.

The successful synthesis of graphene/Fe₃O₄/TiO₂ can be further confirmed by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and power X-ray diffraction (XRD) analysis. The band at 1083 cm⁻¹ and 877.7 cm⁻¹ was attributed to C–O stretching of the epoxy structure in GO, while the bands at 1618.2 cm⁻¹ and 1187.5 cm⁻¹ can be attributed to the stretching vibrations of C–O in asymmetric and symmetric COO⁻ on the graphene/Fe₃O₄ (Fig. S1, ESI†). After coating with polydopamine, the bands at 1083 and 877.7 cm⁻¹ disappear, and many new peaks can be seen in PDA–graphene/Fe₃O₄ (Fig. S1b, ESI†). The peak at 1203 cm⁻¹ was assigned to the C–O stretching of phenolic groups in polydopamine, which indicated that graphene/Fe₃O₄ had been successfully modified by PDA coating. It was obvious that C, O, Fe, Si, N and Ti all appeared on the surface of graphene/Fe₃O₄/TiO₂ composite from XPS patterns (Fig. S2a, ESI†). A C1s peak around 291.7 eV, O1s peak at 530.6 eV, N1s peak at 398.5 eV, Fe2p peak at 710.2 eV and Ti2p peaks at 459.6 and 453.9 eV can be observed. By the deconvolution of C_1s spectrum, three peaks at 284.59, 286.39 and 288.59 eV were identified, which are assigned to the C–C or C–H, C–N or O–C, C=C, and benzene rings respectively (Fig. S2b, ESI†). The XRD results show that the diffraction peaks of graphene/Fe₃O₄/TiO₂ matched well with those of magnetite (JCPDS no. 75-1610) (Fig. S3, ESI†). This suggests the synthetic process did not change the crystalline phase of magnetite. The magnetic properties of graphene/Fe₃O₄/TiO₂ composite were studied using a vibrating sample magnetometer (VSM). The magnetic hysteresis loops show that graphene/Fe₃O₄/TiO₂ composite has a fairly strong magnetization (Fig. S4, ESI†) and can be rapidly separated from the mixture within 1 min in the presence of an external magnet (Fig. S4 inset, ESI†).

The enrichment performance of graphene/Fe₃O₄/TiO₂ composite for phosphopeptides was first examined by the tryptic digest product of β-casein. From the direct analysis of the digest product (20 fmol), it can be seen that only three weak signals of them originating from phosphopeptides could be detected, while the peaks corresponding to nonphosphorylated peptides dominate (Fig. 2a). After the enrichment using graphene/Fe₃O₄/TiO₂ composite, the signals of those at m/z 1981.8, 2061.8, 2432.0, 2556.8, 3042.2 and 3122.1 which represent phosphopeptide residues derived from β-casein¹⁶ can be found (Fig. 2b). Table S1 (ESI†) lists the detailed amino acid sequences of these identified phosphopeptides. When the amount of the digest product decrease to 20 amol and 2 amol, the phosphopeptide peaks at m/z 1981.8, 2061.8, 2556.8 and 3122.1 could still be observed in the mass spectrum after the enrichment using graphene/Fe₃O₄/TiO₂ composite (Fig. 2c and d). As the amount of digest product decreases to 0.2 amol, it is difficult to distinguish the peaks because of the background (Fig. S5, ESI†). The results indicate that graphene/Fe₃O₄/TiO₂ multifunctional composite can specifically extract and completely elute phosphopeptides from the digest solution with high sensitivity.

Fig. 2 MALDI-MS spectra obtained from the tryptic digest of β-casein. (a) Direct analysis of the tryptic digest of β-casein with 20 fmol, and after enrichment by graphene/Fe₃O₄/TiO₂ composite from the tryptic digest of β-casein with 20 fmol (b), 20 amol (c) and 2 amol (d). The peaks of phosphopeptides are marked with #.

The capacity of graphene/Fe₃O₄/TiO₂ composite to selectively trap phosphopeptides was further investigated by analyzing the mixture of tryptic digests of β-casein (20 fmol) and BSA at molar ratio of 1 : 100, 1 : 500. As shown in Fig. S6a (ESI†), nearly no phosphopeptide signal can be observed in the β-casein/BSA tryptic digest mixture at molar ratio of 1 : 100. Because the introduction of BSA dramatically increases the complexity of the sample, the signals of phosphopeptides are severely suppressed by the greater amount of nonphosphopeptides largely originating from BSA. However after the enrichment with graphene/Fe₃O₄/TiO₂ composite, five intensive peaks corresponding to specific phosphopeptides of β-casein are observed. When adjusting β-casein/BSA to 1 : 500 with the concentration of β-casein kept at 20 fmol, after the enrichment, the mass spectra revealed that three signals of phosphopeptides of m/z at 2061.8, 2556.1 and 3122.3 (Fig. S6c, ESI†) can be still observed under the clear background. This proves that graphene/Fe₃O₄/TiO₂ composite possessed the excellent capability for the selective enrichment of phosphopeptides from a complex in the presence of large excess of nonphosphopeptide.

It is demonstrated that the graphene/Fe₃O₄/TiO₂ composite can be applied in the enrichment of phosphopeptides from the tryptic digest of nonfat milk commonly containing α-casein and β-casein. As shown in Fig. 3a, before the enrichment, only a few signals with low intensity from phosphopeptides were identified, which are almost submerged by those of nonphosphopeptides. However, after the enrichment using graphene/Fe₃O₄/TiO₂ composite, eleven phosphopeptides peaks were identified (seven from α-casein and four from β-casein) with good resolution^8a (Fig. 3b) and the corresponding sequences were listed in Table S2 (ESI†). This result demonstrated that graphene/Fe₃O₄/TiO₂ composite can be successfully used for the selective enrichment of phosphopeptides from a very complex real sample.

Fig. 3 MALDI-MS spectra of the tryptic digest of nonfat milk obtained by direct analysis (a), and after enrichment by graphene/Fe₃O₄/TiO₂ composite (b). Peaks of α-casein phosphopeptides are marked with *, and those of β-casein are marked with #.

In summary, a new type of graphene/Fe₃O₄/TiO₂ composite with excellent hydrophilicity and biological compatibility has been synthesized. The multifunctional material was successfully used to highly selectively and sensitively enrich phosphopeptides. Thus, there is promise that the newly synthesized graphene/Fe₃O₄/TiO₂ composite enables a more efficient enrichment of phosphopeptides from a complex peptide mixture in phosphoproteome research.

Acknowledgements

We gratefully appreciate the financial support by the National Basic Research Program of China (no. 2012CB910601), the National Natural Science Foundation of China (no. 20935001, 21275080) and the Research Fund for the Doctoral Program of Higher Education of China (no. 20120031110007).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: The experimental details and additional figures and tables. See DOI: 10.1039/c4ra01573h

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