Synthesis of a hydrophilic maltose functionalized Au NP/PDA/Fe₃O₄-RGO magnetic nanocomposite for the highly specific enrichment of glycopeptides

Abstract

The development of methods to isolate and enrich low-abundance glycopeptides is an important prerequisite for glycoproteomics research. In this study, a hydrophilic maltose functionalized Au nanoparticle (NP)/polydopamine (PDA)/Fe₃O₄-reduced graphene oxide (RGO) nanocomposite has been successfully synthesized in mild conditions. The bioadhesive polydopamine film was prepared by self-polymerization on the surface of Fe₃O₄–graphene oxide, which not only prevents the agglomeration of the graphene sheets and enhances the specific surface area, but also facilitates the Au NP immobilization. A great number of loading Au NPs possess the highly available surface area for the immobilization of the high density of the thiol-terminated maltose via Au–S bonds. The resulting Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposite exhibits excellent environmental stability, good biocompatibility and water dispersibility. Furthermore, the highly loaded Fe₃O₄ NPs make the enrichment very convenient. With all of these advances, the novel Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposite presents selective enrichment of the glycopeptides from a low concentration of horseradish peroxidase tryptic digest (0.1 ng μL⁻¹).

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1 Introduction

As one of the most common post-translational modifications of proteins, glycosylations play important roles in physiological processes, such as immune response, receptor–ligand interactions and signal transduction.^1,2 Anomalous glycosylations are considered to be correlated with numerous diseases, such as cancer,³ neurodegenerative disorders⁴ and other genetic abnormalities.^5,6 Thus, obtaining the information on glycopeptides is significant to understanding the glycoprotein functions in biological pathways and disease development as well as in biomarker discovery.^7,8 Up to now, mass spectrometry (MS) has become an effective tool for analyzing glycopeptides/glycoproteins. However, the direct analysis of low-abundance glycoproteins and glycopeptides extracted from complex biological samples by MS is a great challenge, because the signals of low-abundance glycoproteins and glycopeptides are suppressed by the billion-fold excess quantities of a few highly abundant proteins, making enrichment steps an important prerequisite for successful analysis.^9–11

Over the past decade, several methods for the isolation/enrichment of glycopeptides have been developed, including lectin affinity chromatography,^12,13 hydrazide chemistry,^14,15 boronic acid chemistry^16,17 and hydrophilic interaction chromatography (HILIC).^18,19 Among them, HILIC, which relies on the physical interactions between glycans and the stationary phase, has aroused much interest due to its unbiased enrichment towards different glycopeptides, with remarkable reproducibility and MS compatibility for the separation of polar compounds. However, high selectivity is a great challenge towards glycopeptide enrichment. One of the most promising approaches to solve this issue is the development of novel HILIC materials. In recent years, maltose modified HILIC has attracted more and more attention, due to its superior selectivity and narrower elution window than sepharose, which can be explained by the flexible saccharide chains.^20,21 On the other hand, several HILIC stationary materials have been prepared on silica,^22,23 sepharose²⁴ and magnetic nanoparticles,^25,26 and the aggregation of the particles limits the available surface area of the adsorbent for the efficient enrichment.

Graphene and its derivatives (such as graphene oxide, GO, and reduced graphene oxide, RGO) have attracted a great deal of attention in sample pretreatment due to their ultrahigh specific surface area, high loading capacity, large delocalized π-electron system and hydrophobic interaction.^27–29 However, achieving efficient isolation of the graphene and the targets from the samples has some technological challenges, due to the easy stacking of graphene sheets and the relatively low density of the surface functional groups. Many efforts have been devoted to overcome these challenges. Depositing nanoparticles on GO sheets can also prevent graphene nanosheets from restacking and thus endows new functionality to this 2D carbon nanomaterial.³⁰ As we know, iron oxide nanoparticles (Fe₃O₄ NPs) have the properties of good hydrophilicity, biocompatibility and magnetic responsibility^31–33 which will facilitate the separation process in comparison with conventional approaches. The hybrid of graphene and Fe₃O₄ NPs could effectively broaden the applicability.³⁴ On the other hand, dopamine commonly known as a neuroendocrine transmitter and a unique molecule which mimics the adhesive proteins, has been found to be able to polymerize into a unique hydrophilic polydopamine (PDA) coating on organic and inorganic surfaces under mild conditions.^35–38 Yang et al. reported that PDA as an adhesive coating on graphene sheets prevents the agglomeration of the graphene sheets and enhances the specific surface area.³⁹ Meanwhile, The PDA coating remains stable even if in a harsh environment such as a strong acid solution, so it can protect the magnetic particles from etching in acid solution. Meanwhile, there are strong adhesive forces and electrostatic interactions between the PDA and the gold NPs. As a result, the gold NPs could be fabricated on the surface of the PDA.^40,41

In this work, we report a facile approach to synthesize a novel hydrophilic magnetic composite Au NP-maltose/PDA/Fe₃O₄-RGO. This nanocomposite has the following merits: (1) the large specific surface area of reduced graphene oxide (RGO) offers a higher capacity for loading the Au NPs and thus possesses the highly available surface area for the immobilization of maltose. (2) The hydrophilic PDA layer on the magnetic graphene exhibits excellent environmental stability, good biocompatibility and good water dispersibility. (3) Lots of thiol-terminated maltose molecules were assembled onto the Au NPs via Au–S bonds, improving its hydrophilicity. Further, the long organic chains, bridging the Au NPs and the thiol-terminated maltose could suppress the non-specific adsorption. (4) The highly loaded Fe₃O₄ NPs make the enrichment very convenient. The resulting nanocomposite was successfully applied to the highly selective capture of glycopeptides from complex biosamples.

2 Experimental

2.1 Materials

Horseradish peroxidase (HRP) (MW ∼ 44 kDa), bovine serum albumin (BSA), DL-dithiothreitol (DTT), urea and High Performance Liquid Chromatography (HPLC)-grade acetonitrile (ACN) were purchased from Sigma-Aldrich (USA). Graphene oxide (GO) was obtained from XF NANO (China). 2,5-Dihydroxybenzoic acid (DHB), iodoacetamide (IAA), dopamine hydrochloride and hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂O) were obtained from Alfa Aesar (USA). Amberlite IR120 was obtained from J&K (China). Urea, iron(III) chloride hexahydrate (FeCl₃·6H₂O), anhydrous sodium acetate (NaAc), ethylene glycol (EG), sodium borohydride (NaBH₄), ammonium bicarbonate (NH₄HCO₃), formic acid (FA), D-maltose, acetic anhydride (Ac₂O), potassium thioglycolate (KSAc), sodium hydroxide (NaOH), para-toluensulfonyl chloride (TsCl), tetraethylene glycol, anhydrous sodium sulfate (Na₂SO₄), dichloromethane (CH₂Cl₂), methanol, ethanol, sodium, n-hexane, petroleum ether and ethyl acetate were purchased from the Tianjin Chemical Reagent Company (Tianjin, China). Deionized water (18.25 MΩ cm) was prepared with a Milli-Q water purification system (Millipore, Milford, MA, USA).

2.2 Preparation of Fe₃O₄–GO nanocomposites

The Fe₃O₄–GO nanocomposites were synthesized with a solvothermal method.⁴² Typically, GO (48.7 mg), NaAc (1.36 g) and FeCl₃·H₂O (152 mg) were dissolved in EG (30 mL) under sonication to give a homogenous solution which was then poured into a Teflon-lined stainless-steel autoclave (50 mL). After reaction at 180 °C for 16 h, the Fe₃O₄–GO nanocomposites were washed with deionized water and ethanol several times, and were then dried at 50 °C overnight.

2.3 Preparation of PDA-coated Fe₃O₄–GO nanocomposites (PDA/Fe₃O₄-RGO)

The dopamine hydrochloride (30 mg) was added into a solution of the Fe₃O₄–GO (40 mg) in 10 mM Tris buffer (pH 8.5). The reaction solution was sonicated for 10 min, and stirred vigorously at 60 °C for 24 h. The product of PDA/Fe₃O₄-RGO was washed with deionized water and ethanol thrice, and was then dried at 50 °C overnight.

2.4 Preparation of Au NP/Fe₃O₄-RGO and Au NP/PDA/Fe₃O₄-RGO nanocomposites

Into a solution of the Fe₃O₄-RGO (or PDA/Fe₃O₄-RGO) nanocomposites (40 mg) in deionized water (60 mL) was added 24 mL HAuCl₄·3H₂O (1 mg mL⁻¹). The solution was sonicated for 5 min, and stirred for 2 h at room temperature. Then freshly prepared NaBH₄ (10 mg mL⁻¹) was added and stirred continuously for another 2 h.⁴³ The Au NP/Fe₃O₄-RGO or Au NP/PDA/Fe₃O₄-RGO nanocomposites were washed with deionized water and ethanol thrice, and were then dried at 50 °C for 3 h.

2.5 Preparation of thiol-terminated maltose (3)

The reagents and conditions for the preparation of the thiol-terminated maltose (3) are shown in Scheme 2. The synthesis procedure for compounds 1–3 is in the following text.

Scheme 1 Schematic illustration of the fabrication of the hydrophilic magnetic Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites.

Scheme 2 Synthesis of the thiol-terminated maltose 3. Reagents and conditions: (i) TsCl, NaOH, THF, (0 °C);⁴⁵ (ii) BF₃·Et₂O, CH₂Cl₂; (iii) (a). KSAc, 2-butanone, (b). 1 M NaOMe/MeOH, MeOH.^46,47

2-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)ethyl p-tosylate (1). Into a solution of tetraethylene glycol (21.95 g, 113 mmol) in CH₂Cl₂ (4 mL) was added an aqueous NaOH (0.69 g, 17.13 mmol) solution (4 mL) dropwise at 0 °C under a nitrogen atmosphere, and then a THF solution (20 mL) of p-toluenesulfonyl chloride (2.08 g, 10.93 mmol) was added dropwise slowly. The resulting mixture was allowed to stir at 0 °C for 2 h. The reaction mixture was poured onto ice H₂O (70 mL), and the solvent was concentrated to ∼2/3 volume under reduced pressure. The remaining solution was extracted with CH₂Cl₂ (3 × 60 mL), and the combined organic layers were washed with brine (40 mL), and dried over Na₂SO₄. The crude mixture was filtered, concentrated and purified by silica gel chromatography (ethyl acetate/petroleum ether, 30 : 70 to 70 : 30) to give the product as a pale yellow oil (5.53 g, 20.4%).

2-(2-{2-[2-(2-Tosyloxy-ethoxy)-ethoxy]-ethoxy}-ethyl)2,3,4,6-tetra-O-β-D-glucopyranosyl-(1→4)-2,3,6tri-O-acetyl-β-D-glucopyranoside (2). Into a solution of octa-O-acetyl-D-maltopyranose (3.72 g, 5.5 mmol) and 1 (1.58 g, 6.0 mmol) was added BF₃·Et₂O (2.4 mL, 8.4 mmol) at 0 °C under a nitrogen atmosphere. After stirring for 24 h at room temperature, the reaction mixture was diluted with NaHCO₃, extracted with CH₂Cl₂, washed with brine, and then dried over Na₂SO₄. After filtration of the crude mixture, the crude product was concentrated and purified by silica gel chromatography (ethyl acetate/hexane 50 : 50–80 : 20) to give the product as a pale yellow oil (1.38 g, 26%).

2-(2-{2-[2-(2-Thiol-ethoxy)-ethoxy]-ethoxy}-ethyl)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside (3). Into a solution of 2 (1.5 g, 1.55 mmol) in 2-butanone (30 mL) was added potassium thioacetate (0.35 g, 3.1 mmol). The resulting mixture was stirred under reflux for 2 h. The solvent was evaporated under reduced pressure, then the residue was dissolved in CH₂Cl₂ (100 mL) and washed with brine. The organic layer was dried over Na₂SO₄ and evaporated under reduced pressure. After purification by silica gel chromatography (ethyl acetate/petroleum ether 30 : 30–50 : 50), the residue was solvated into dry MeOH, and NaOMe/MeOH (0.84 M, 1 eq.) was added at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 3 h. The reaction mixture was neutralized by Amberlite IR 120 H until pH 6, after filtration the solvent was evaporated under reduced pressure to give the product as a pale brown oil. ¹H NMR (400 M Hz, DMSO, δ, ppm): 5.53–5.48 (m, 2H), 5.14 (d, J = 4.8 Hz, 1H), 5.00 (d, J = 3.7 Hz, 1H), 4.94 (m, 2H), 4.54 (m, 2H), 4.20 (d, J = 7.7 Hz, 1H), 3.89–3.85 (m, 1H), 3.72–3.42 (m, 19H), 3.35–3.16 (m, 4H), 3.09–3.00 (m, 2H), 2.90 (t, J = 6.4 Hz, 1H), 2.61 (t, J = 6.68 Hz, 1H), 2.51–2.50 (m, 1H). HR MS (ESI): calcd for C₂₀H₃₈O₁₄S [M + Na]⁺ 557.1874, found 557.1876.

2.6 Preparation of Au NP-maltose/Fe₃O₄-RGO and Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites

Into a solution of Au NP/Fe₃O₄-RGO or Au NP/PDA/Fe₃O₄-RGO nanocomposites (30 mg) in ethanol (60 mL) was added thiol-terminated maltose (300 mg). The solution was sonicated for 5 min, and stirred at room temperature for 24 h.⁴⁴ With a magnetic separation technique, the Au NP-maltose/Fe₃O₄-RGO or Au NP-maltose/PDA/Fe₃O₄-RGO composites were washed with ethanol thrice, and then dried at 50 °C for 3 h.

2.7 Characterization

The morphology and structure of the synthesized magnetic graphene composites were evaluated using a Tecnai G2T2 S-TWIN transmission electron microscope (TEM). Samples for TEM were prepared by placing a drop of the dilute particles of a solution in the ethanol solvent on a copper grid. The infrared spectra were recorded on a Nicolet AVATAR-360 Fourier transform infrared (FT-IR) spectrometer. After vacuum drying, the samples were thoroughly mixed with KBr (the weight ratio of sample/KBr was 1%) in a mortar, and then the fine powder was pressed into a pellet. The identification of the crystalline phase was performed on a Rigaku D/max/2500v/pc (Japan) X-ray diffractometer with a Cu Kα source. The 2θ angles probed were from 3° to 80° at a rate of 4° min⁻¹. The hydrophilicity was evaluated with a contact angle analyzer JCY-1 (Fangrui, China). The magnetic properties were analyzed with a vibrating sample magnetometer (VSM) (LDJ 9600-1, USA).

2.8 Enrichment of glycopeptides

The tryptic digest of HRP was prepared in the following way. Typically, HRP was dissolved in NH₄HCO₃ (50 mM, pH 8.0) to the final concentration (1 mg mL⁻¹) and denatured at 100 °C for 5 min. Then, trypsin was added into the solution at an enzyme/substrate ratio of 1 : 50 (w/w) which was then incubated at 37 °C for 16 h. The digestion of the BSA was performed by incubating a mixture of 1 mg BSA, 100 μL of 8 M urea and 20 μL of a 50 mM NH₄HCO₃ solution (pH 8.0) at 56 °C for 1 h, and then by mixing 20 μL of 200 mM IAA at 37 °C in the dark for 30 min, and then by adding a 860 μL NH₄HCO₃ solution and the trypsin into the mixture at a enzyme/protein ratio of 1 : 50 (w/w) for 12 h at 37 °C. After the addition of formic acid (5 μL) to quench the reaction, the tryptic digestion was stored at −20 °C until use.

Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites (1 mg) were added into the HRP digests or into a mixed sample (HRP, BSA digests) (400 μL) dissolved in CH₃CN/H₂O/FA (80 : 19.9 : 0.1) with shaking for 1 h at RT, it was then washed thrice with the same solution (CH₃CN/H₂O/FA = 88 : 19.9 : 0.1, 3 × 400 μL, 10 min) to remove the nonspecifically adsorbed peptides. A solution of CH₃CN/H₂O/FA (10 : 89 : 1, 15 μL) was added to release the glycopeptides at RT for 30 min. The elutions were analyzed by MALDI-TOF MS.

2.9 MALDI-TOF MS analysis

MALDI-TOF MS analysis was performed on a Autoflex III LRF 200-CID TOF/TOF mass spectrometer (Bruker Daltonics, Bermen, Germany). The DHB matrix was prepared by adding 2,5-dihydroxybenzonic acid (DHB, 12 mg) into a solution of CH₃CN/H₂O/FA (30 : 69 : 1). Equivalent amounts (1 μL) of the elute and DHB matrix were sequentially dropped onto the MALDI plate for MS analysis. The MALDI mass spectra were obtained in the positive-ion reflector mode.

3 Results and discussion

3.1 Preparation and characterization of Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites

The stepwise fabrication of the Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites is illustrated in Scheme 1. Firstly, the magnetic graphene oxide (Fe₃O₄–GO) was synthesized with a solvothermal method.⁴² The GO highly loaded with Fe₃O₄ NPs was hydrothermally produced from the reduction reaction between FeCl₃ and ethylene glycol in the presence of GO. Secondly, dopamine was oxidized to form the PDA layer on the surface the Fe₃O₄–GO. This step is vital, because the PDA layer not only protects the stability of the magnetic nanoparticles and improves the hydrophilicity of graphene, but also maintains the good dispersion of the gold nanoparticles (Au NPs) on the surface of the RGO for further functionalization. Thirdly, Au NPs were loaded onto the surface of the PDA through the sodium borohydride reduction. Finally, the highly dense thiol-terminated maltose 3 was assembled onto the surface of the Au NPs with Au–S bonds to form the Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites.

The sizes and morphology of the products of the Fe₃O₄–GO, PDA/Fe₃O₄-RGO, Au NP/PDA/Fe₃O₄-RGO and Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites were characterized by TEM. As shown in Fig. 1a, there are a lot of Fe₃O₄ NPs having an average diameter of approximately 200 nm which were dispersed on the surface of the GO nanosheets. After the formation of the PDA coating by self-polymerization, the morphology of the PDA/Fe₃O₄-RGO remains as the inherent Fe₃O₄–GO sheets with the relatively thick coating (Fig. 1b). It can be seen from Fig. 1c, that there is a great amount of Au NPs with an average diameter of ∼5 nm obviously loaded onto the surface of the PDA/Fe₃O₄-RGO sheets by direct reduction of HAuCl₄ using sodium borohydride as the reducing agent. Meanwhile, the Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites indicated that the modification of the thiol-terminated maltose did not cause agglomeration (Fig. 1d).

Fig. 1 TEM images of the Fe₃O₄–GO (a); PDA/Fe₃O₄-RGO (b); Au NP/PDA/Fe₃O₄-RGO (c); Au NP-maltose/PDA/Fe₃O₄-RGO (d) nanocomposites.

Fourier transform infrared (FT-IR) spectroscopy in the range of 4000–450 cm⁻¹ was employed to monitor the stepwise synthesis of the Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites. As shown in Fig. 2a, the peaks at 1726 cm⁻¹, 1217 cm⁻¹ and 1051 cm⁻¹ were ascribed to the stretching vibration of C=O bonds, to the C–O and C–O–C bonds of the epoxy structures and to the –COOH groups in GO, respectively, while the band of 1623 cm⁻¹ is assigned to the C=C stretching vibration of graphene. The peaks of Fe₃O₄–GO (Fig. 2b) at 590 cm⁻¹ and 1560 cm⁻¹ were assigned to the vibration the Fe–O bonds and to the anti-symmetrical vibration of COO⁻. After coating the dopamine, the weak peak of PDA/Fe₃O₄-RGO (Fig. 2c) at 1457 cm⁻¹ was substituted by the C–C vibrations of the benzene ring. Compared to Fe₃O₄–GO, the C=O peak at 1720 cm⁻¹ disappeared. It indicated that the Fe₃O₄–GO was reduced to PDA/Fe₃O₄-RGO by the modification of PDA. Meanwhile, the characteristic peaks in Fig. 2c–e were virtually identical, which demonstrated that the framework of the PDA/Fe₃O₄-RGO remains unchanged.

Fig. 2 FT-IR spectra of GO (a), and of the Fe₃O₄–GO (b), PDA/Fe₃O₄-RGO (c), Au NP/PDA/Fe₃O₄-RGO (d), and Au NP-maltose/PDA/Fe₃O₄-RGO (e) nanocomposites and dopamine (f).

The crystalline property of the Fe₃O₄–GO, PDA/Fe₃O₄-RGO, Au NP/PDA/Fe₃O₄-RGO and Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites was characterized by X-ray diffraction (XRD). As shown in Fig. 3, in the 2θ range of 3–80°, the characteristic peaks for Fe₃O₄ (2θ = 30.1°, 35.5°, 43.1°, 53.4°, 57.1°, 62.7°) at the corresponding 2θ values are indexed as (220), (311), (400), (422), (511) and (440), respectively, which can be indexed to the face centered-cubic phase of Fe₃O₄ (JCPDS card no. 19-629). Meanwhile, the diffraction peaks at 38.1°, 44.3°, 64.6°, 77.4° of Au NP-maltose/PDA/Fe₃O₄–GO are observed, which represent the Bragg reflections from the (111), (200), (220) and (311) planes of Au (JCPDS card no. 01-1174), showing clearly the existence of the Au NPs in the Au NP/PDA/Fe₃O₄-RGO and Au NPs-maltose/PDA/Fe₃O₄-RGO composites (Fig. 3c and b). The magnetic properties of the synthesized nanocomposites were studied using a vibrating sample magnetometer (VSM) at room temperature. As shown in Fig. 4, the magnetization saturation (M_s) values of the Fe₃O₄–GO, PDA/Fe₃O₄-RGO, Au NP/PDA/Fe₃O₄-RGO and Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites were 40.09, 21.73, 16.69 and 16.35 emu g⁻¹, respectively. The decrease in the M_s of PDA/Fe₃O₄-RGO in comparison with Fe₃O₄–GO is attributed to the increased mass of the self-assembled polydopamine layer on the surface of Fe₃O₄–GO. The M_s value of Au NP/PDA/Fe₃O₄-RGO was lower than that of PDA/Fe₃O₄-RGO, due to the modification of the Au NPs on the surface of PDA/Fe₃O₄-RGO. Nevertheless, after thiol-terminated maltose was functionalized onto the surface of the Au NPs, the M_s of the Au NP-maltose/PDA/Fe₃O₄-RGO remained about the same. And the magnetization value was sufficient to accomplish the fast and efficient separation with an external magnetic field. The resulting nanocomposite materials also show a fast response to the applied magnetic field (2000 Oe). After dispersing the Au NP-maltose/PDA/Fe₃O₄-RGO composites in water by shaking and sonication, they can be easily separated within 30 s with the help of the magnetic field (Fig. 4 inset). This suggests that the novel nanocomposite materials possess excellent magnetic properties and redispersibility, which is advantageous for application in bioseparation.

Fig. 3 XRD patterns of the Fe₃O₄–GO (a), PDA/Fe₃O₄-RGO (b), Au NP/PDA/Fe₃O₄-RGO (c) and Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites.

Fig. 4 Hysteresis loops of the magnetic Fe₃O₄–GO (a), PDA/Fe₃O₄-RGO (b), Au NP/PDA/Fe₃O₄-RGO (c) and Au NP-maltose PDA/Fe₃O₄-RGO (d) nanocomposites. The inset picture shows the dispersion, and magnetic field induced separation, of the Au NP-maltose/PDA/Fe₃O₄-RGO composites.

To demonstrate the influence of the hydrophilicity of the nanocomposites on the enrichment, we have added the contact angle measurements for the composite materials in each step. Deionized water was used as a probe liquid. When deionized water was put on the membrane surface, the water contact angle was measured immediately and was averaged from three points. Generally, a smaller contact angle indicates better hydrophilicity of the materials. The contact angle of the Fe₃O₄–GO (Fig. 5a), PDA/Fe₃O₄-RGO (Fig. 5b) and Au NP-maltose/PDA/Fe₃O₄-RGO (Fig. 5c) nanocomposites were 52.95°, 50.92° and 44.31° separately, implying an enhancement in hydrophilicity after modification of the PDA and maltose.

Fig. 5 Shape of the water droplets on the Fe₃O₄–GO (a), PDA/Fe₃O₄-RGO and Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites.

3.2 Selective enrichment of glycopeptides

The selective isolation and enrichment of glycopeptides is based on the hydrophilic interaction between the hydrophilic maltose molecules and the glycopeptides. To demonstrate the enhanced enrichment of the glycopeptides due to the hydrophilicity of the PDA and the assembly of maltose on the surface of the Au NPs, the Fe₃O₄–GO, Au NP-maltose/Fe₃O₄-RGO, Au NP/PDA/Fe₃O₄-RGO and Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites were used to enrich the glycopeptides from the HRP digests (Fig. 6). For the direct analysis of the HRP digest (10 ng μL⁻¹) without enrichment, only one peak (H7) could be observed, due to the suppression of the non-glycopeptides (Fig. 6A). The Fe₃O₄–GO nanocomposite without the modification of hydrophilic PDA and maltose showed no enrichment (Fig. 6B1). If the Au nanoparticles are immobilized on the Fe₃O₄–GO directly and are followed by hydrophilic maltose functionalization, then no glycopeptide signals were detected (Fig. 6B2). After the modification of polydopamine on the graphene oxide with reference supporting, two glycosylated peptides were assigned without assembly of the maltose (Fig. 6C).⁴² Thankfully, after enrichment with the Au NP-maltose/PDA/Fe₃O₄–GO nanocomposites, non-glycosylated signals were removed effectively and the additional 8 glycosylated peptides could be identified except for peak H7. Moreover, the signal-to-noise (S/N) ratio of the glycopeptides was obviously enhanced (Fig. 6D). The detailed structure of the glycopeptides in Fig. 6D is listed in Table 1. The results indicate that the modification of the PDA layer and maltose on the nanocomposite synergically contributed to improving its hydrophilicity which enhanced the selectivity of the glycopeptide enrichment.

Fig. 6 MALDI mass spectra of the tryptic digest of 10 ng μL⁻¹ HRP (A) without and with enrichment by Fe₃O₄–GO (B1), Au NP-maltose/Fe₃O₄-RGO (B2), Au NP/PDA/Fe₃O₄-RGO (C) and Au NP-maltose/PDA/Fe₃O₄-RGO (D) nanocomposites. The peaks of the glycopeptide fragments are marked with H.

Table 1 Detailed information of the glycopeptides enriched by the Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites from HRP digests¹⁰

Peak number	Observed m/z	Glycan composition	Amino acid sequence^a
a The N-glycosylation sites are marked with N#. GlcNAc = N-acetylglucosamine, Fuc = fructose, Man = mannose, Xyl = xylose.
H1	2321.0	Man2GlcNAc2	MGN#ITPLTGTQGQIR
H2	2591.0	XylMan3FucGlcNAc2	PTLN#TTYLQTLR
H3	2850.0	FucGlcNAc	GLIQSDQELFSSPN#ATDTIPLVR
H4	3087.8	XylMan3FucGlcNAc2	GLCPLNGN#LSALVDFDLR
H5	3323.6	XylMan3FucGlcNAc2	QLTPTFYDNSCPN#VSNIVR
H6	3606.3	XylMan3FucGlcNAc2	NQCRGLCPLNGN#LSALVDFDLR
H7	3671.9	XylMan3FucGlcNAc2	GLIQSDQELFSSPN#ATDTIPLVR
H8	4223.3	XylMan3FucGlcNAc2	QLTPTFYDNSC(AAVESACPR)PN#VSNIVR
H9	4983.8	XylMan3FucGlcNAc2	LYN#FSNTGLPDPTLN#TTYLQTLR
		XylMan3FucGlcNAc2

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The sensitivity of the above prepared nanocomposites for glycopeptides was detected with different concentrations of the HRP tryptic digest. As shown in Fig. 7, the glycopeptide peaks H1–H9 detected in the elutions of the 10 ng μL⁻¹ HRP digests did not appear except for the peaks H7 and H9 along with the decrease of the concentration of the HRP digest. Nevertheless, the two glycopeptide peaks (H7, H9) could be still observed after enrichment with the Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites from the HRP digests (0.1 ng μL⁻¹). The results indicate that the hydrophilic Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites can specifically extract glycopeptides from the HRP digest solution with high sensitivity.

Fig. 7 MALDI mass spectra of the tryptic digest of (A) 5, (B) 1, (C) 0.2, (D) 0.1 ng μL⁻¹ HRP after enrichment with Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites.

The performance of the composite material for glycopeptide enrichment from a complicated peptide mixture or biological samples was carried out. Fig. 8A shows the direct analysis of the tryptic digest mixture of HRP and BSA (1 : 10, mass ratio), the glycosylated peptide signals were suppressed absolutely. In contrast, after enrichment by the Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites (Fig. 8B), 2 peaks (H7, H9) could be clearly detected. The results indicate that the Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites have a high enrichment efficiency and selectivity for the glycopeptides from a complicated biosample.

Fig. 8 MALDI mass spectra of the tryptic digest mixture of HRP and BSA (1 : 10, mass ratio) (A) without and with enrichment by the Au NP-maltose/PDA/Fe₃O₄-RGO nanocomposites (B).

4 Conclusions

In conclusion, a novel hydrophilic thiol-terminated maltose-functionalized Au NP/PDA/Fe₃O₄-RGO nanocomposite was synthesized under mild conditions. Due to the enhanced hydrophilicity by the PDA layer and the suppressed nonspecific adsorption of non-glycosylated peptides by the organic chains bridging the Au NPs and the maltose, the resulting nanocomposite exhibited high selectivity and detection sensitivity in the enrichment of glycopeptides from complex samples without any pretreatment. The novel nanocomposites are expected to become a promising strategy to design an efficient and sensitive tool for glycoproteomic analysis.

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 21275080, 21475067) and the Research Fund for the Doctoral Program of Higher Education of China (no. 20120031110007) and the National Natural Science Foundation of Tianjin (no. 15JCYBJC20600).

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