Click Synthesis of Hydrophilic Maltose-Functionalized Iron Oxide

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Click synthesis of hydrophilic maltose-functionalized iron oxide magnetic nanoparticles based on dopamine anchors for highly selective enrichment of glycopeptides Changfen Bi, Yingran Zhao, Lijin Shen, Kai Zhang, Xi-Wen He, Langxing Chen, and Yu-Kui Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b06991 • Publication Date (Web): 19 Oct 2015 Downloaded from http://pubs.acs.org on October 27, 2015

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ACS Applied Materials & Interfaces

Click synthesis of hydrophilic maltose-functionalized iron oxide magnetic nanoparticles based on dopamine anchors for highly selective enrichment of glycopeptides Changfen Bi,†,‡ Yingran Zhao, †,‡ Lijin Shen, § Kai Zhang, § Xiwen He, †,‡ Langxing Chen*, †,‡ and Yukui Zhang†,‡,#

†

Research Center for Analytical Sciences, College of Chemistry, Tianjin Key Laboratory of Biosensing

and Molecular Recognition, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China Fax: (+86) 22-2350-2458, E-mail: [email protected] ‡

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071,

China §

Department of Biochemistry and Molecular Biology &Tianjin Key Laboratory of Medical Epigenetics,

Tianjin Medical University, Tianjin, 300070, China #

Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, China

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ABSTRACT: The development of methods to isolate and enrich low-abundance glycopeptides from biological sample is crucial to glycoproteomics. Herein, we present an easy and one-step surface modification strategy to prepare hydrophilic maltose functionalized Fe3O4 nanoparticles (NPs). Firstly, based on the chelation of the catechol ligand with iron atoms, azido-terminated dopamine (DA) derivative was assembled on the surface of magnetic Fe3O4 nanoparticles by sonication. Secondly, the hydrophilic maltose-functionalized Fe3O4 (Fe3O4-DA-Maltose) NPs were obtained via copper (I)catalyzed azide-alkyne cycloaddition (click chemistry). The morphology, structure and composition of Fe3O4-DA-Maltose NPs were investigated by Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), X-ray powder diffraction (XRD),

X-ray photoelectron

spectrometer (XPS) and vibrating sample magnetometer (VSM). Meanwhile, hydrophilicity of the obtained NPs was evaluated by water contact angle measurement. The hydrophilic Fe3O4-DA-Maltose NPs were applied in isolation and enrichment of glycopeptides from horseradish peroxidase (HRP), immunoglobulin (IgG) digests. The MALDI-TOF mass spectrometric analysis indicated that the novel NPs exhibited high detection sensitivity in enrichment from HRP digests at concentration as low as 0.05 ng µL-1, a large binding capacity up to 43 mg g-1 and good recovery for glycopeptides enrichment (85% ~ 110%). Moreover, the Fe3O4-DA-Maltose NPs were applied to enrich glycopeptides from human renal mesangial cells (HRMC) for identification of N-glycosylation sites. Finally we identified 115 different N-linked glycopeptide, representing 93 gene products and 124 glycosylation sites in HRMC.

KEYWORDS: Fe3O4 nanoparticles; Dopamine; Copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC); Maltose; Glycopeptides; Enrichment


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1. Introduction Glycosylation, as one of the most complicated post- translational modifications of proteins, is involved in a variety of biological activities,1 such as protein folding and transport, immune regulation, cell signal transduction.2-4 Aberrant glycosylation has been associated with numerous diseases, such as diabetes, cardiovascular disease, neurodegenerative diseases and cancer.5-7 Therefore, characterization of glycoprotein structures, especially glycosylation site occupancy and glycan heterogeneity at each glycosite, is significant for diagnosis and pathology.8-10 Currently, mass spectrometry (MS) based techniques have been the primary analytical tools for protein glycosylation.11,12 However, owing to the low abundance and poor ionization of glycopeptides, MS signal is suppressed when subjected directly to mass spectrometric analysis.13 Therefore, an effective enrichment of glycopeptides prior to MS analysis becomes imperative.14 To overcome these issues, several strategies for the enrichment of glycopeptides have been developed, including lectin affinity chromatography,15,16 hydrazide chemistry,17-19 boronic acid chemistry20,21 and hydrophilic interaction chromatography (HILIC).22-28 In recent years, HILIC has aroused much attention for glycopeptides enrichment by utilizing the hydrophilicity of the analyte,29,30 due to its broad glycan specificity, reproducibility and MS compatibility.31,32 Magnetic nanoparticles (MNPs), especially, iron oxide Fe3O4 and γ-Fe2O3 have attracted considerable attention in recent years, due to their magnetic responsibility, nontoxicity, biocompatibility,33,34 and their broad range of application in the fields of drug delivery,35 magnetic resonance imaging contrast enhancement,36 cell and proteins guidance/separation,37,38 and proteomics analysis.39 However MNPs are easy to aggregate, surface unstable, which limits their applications. Therefore, the effective surface functionalization is essential for the applications of MNPs. As we known, Fe3O4 NPs have good hydrophilicity and numerous Fe3+/Fe2+ binding sites on the surface. Therefore, it is an effective route that the target group is assembled directly on the surface via metal-organic group coordination. Since the biocompatibility of dopamine and the robust binding of the catechol unit to iron oxide, the


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coordination of dopamine ligands may be functionalized in an easy, straightforward fashion to the surface of iron oxide NPs. After Xu et al. firstly used dopamine derivative as the surface anchor for Fe3O4 NPs,40 some dopamine-based ligands like trimethoprim, glutathione (GSH), nitro-, and azido group linking dopamine as a robust and versatile anchor on the surface of Fe3O4 NPs.41-43 The primary amine of dopamine-coated MNPs allows for post-functionalization of dopamine by connecting other functional groups with amine,44,45 which provided more options for the surface of modification of iron oxide MNPs for biomedical applications. Recently, some articles reported assembly of hydrophilic carbohydrates on the surface MNPs for enrichment of glycopeptides.46-48 However, they usually need the multistep modification to obtain carbohydrates-coated MNPs, which weakens the operability. Therefore, it is worth exploiting new approaches to functionalize MNPs to simplify the procedure and ensure the hydrophilicity of NPs simultaneously. In this work, we present an easy and one-step surface modification strategy to prepare hydrophilic maltose functionalized Fe3O4 MNPs (Scheme 1). The azido-terminated dopamine derivative as a robust anchor functionalized the surface of Fe3O4 MNPs, dopamine bearing azide serves as the reactive point to connect the alkyne-terminated hydrophilic maltose via Cu (I)-catalyzed azide-alkyne cycloaddition (CuAAC).49-51 The obtained maltose-functionalized Fe3O4 MNPs exhibited highly selective isolation and enrichment of glycopeptides from the complex biosamples. /Scheme 1/ 2. Experimental Section 2.1. Materials. Horseradish peroxidase (HRP), immunoglobulin G (IgG), peptide-N-glycosidase (PNGase F), bovine serum albumin (BSA) and HPLC-grade acetonitrile (ACN) were purchased from Sigma-Aldrich (USA). Dithiothreitol (DTT) and iodoaceamide (IAA) were purchased from Solarbio (China). Dopamine hydrochloride (DA) and L-ascorbic acid sodium salt were obtained from Alfa Aesar ACS Paragon Plus Environment

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(USA). Trifluoroacetic acid (TFA), trichloroacetic acid (TCA), Amberlite IR 120 and 2-bromoethanol were purchased from J&K (China). 2,5-Dihydroxybenzoic acid (DHB) was obtained from TCI (Japan). Trypsin was from Sangon Biotech Co. Led. (China). 4-Dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide were obtained from Shanghai Medpep. Co. (China). Iron(II) chloride tetrahydrate (FeCl2·4H2O), Iron (III) chloride hexahydrate (FeCl3·6H2O), sodium azide (NaN3), pentanoic anhydride, D-maltose, tetraethylene glycol, acetic anhydride (Ac2O), sodium hydride (NaH), anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), ammonium bicarbonate (NH4HCO3), boron fluoride ethyl ether (BF3·Et2O), propargyl bromide and other analytical grade reagents were purchased from Tianjin Chemical Reagent Company (China). Deionized water (18.25 MΩ cm) was prepared with a Milli-Q water purification system (Millipore, Milford, MA, USA). 2.2. Characterization. The transmission electron microscope (TEM) images were collected on a Tecnai G2 T2 S-TWIN transmission electron microscope. Fourier transform infrared (FT-IR) spectra (4000- 400 cm-1) in KBr were recorded using the BRUKER TENSOR 27 Fourier transform infrared spectrophotometer. The crystal structure of the NPs was performed on a Rigaku (Japan) D/max/2500v/pc with nickel-filtered Cu Kα source. The XRD patterns were collected in the range 3°< 2θ< 80° at a scan rate of 4.0°/min. The X-ray photoelectron spectra were obtained on a Shimadzu (Japan) Kratos AXIS Ultra DLD X-ray photoelectron spectrometer (XPS) with an Mg Ka anode (15 kV, 400 W) at a takeoff angle of 45°. The source X-rays were not filtered and the instrument was calibrated against the C1s band at 285 eV. The magnetic properties were analyzed with a LDJ9600-1 (USA) vibrating sample magnetometer. The hydrophilicity of NPs was evaluated with contact angle analyzer JCY-1 (Fangrui, China). Zeta potential of the nanoparticles at a different pH values were measured by Malvern Zetasizer Nano-2S&MPT-2 (Worcestershine, UK) at room temperature. The matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) experiments were performed on a Autoflexш LRF 200-CID TOF/TOF mass spectrometer (BrukerDaltonics, Bermen, Germany). The


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DHB matrix was prepared by adding 2, 5-dihydroxybenzonic acid (DHB, 25 mg mL-1) into a solution of CH3CN/H2O/TFA (80: 19: 1). Equivalent amounts (1 µL) of the eluate and DHB matrix were sequentially dropped onto the MALDI plate for MS analysis. The MALDI mass spectra were obtained in positive ion reflector mode. 2.3. Synthesis of Fe3O4 NPs. Fe3O4 NPs were synthesized by co-precipitation method.52 FeCl3·6H2O (2.36 g) was dissolved into ultrapure water (40 mL) in a three-neck flask under nitrogen atmosphere. After 10 minutes, NH3·H2O (5 mL) and FeCl2·4H2O (0.86 g) were added in quickly and the resulting mixture was allowed to stir at 80 °C for 30 min. Under an external magnetic field, the Fe3O4 NPs were separated from the solution, washed with ultrapure water until the supernatant was neutral, washed with ethanol, then dried at 50 °C overnight. 2.4. Synthesis of dopamine-N3 (1). The synthesis of dopamine-N3 is in the following. Dopamine hydrochloride (1.53 g, 8.1 mmol) was dissolved in MeOH (30 mL), and Et3N (0.81 g, 8.1 mmol) was added. A solution of 2-azidoethyl-2,5-dioxopyrrolidin-1-yl glutarate53,54 (2.41 g, 8.1 mmol) in MeOH (20 mL) was added dropwise to the first solution. The reaction was stirred vigorously for 48h. The solvent was evaporated under reduced pressure, and the residue was dissolved in CH2Cl2 (100 mL). The organic phase was washed with HCl (0.5 M, 10.8 mL) and dried over Na2SO4. The crude mixture was filtered, concentrated and purified by column chromatography (SiO2, CH2Cl2: methanol 10:1), to give the product a light brown solid. 1

H NMR (300 M Hz, CDCl3, δ, ppm) 7.72 (br, 1H, ArH), 6.78 (d, 1H, ArH), 6.72 (s, 1H, ArH), 6.51

(d, 1H, ArH), 4.21 (t, 2H, CH2-O-CO), 3.41 (m, 4H, CH2-NH-, CH2-N3), 2.65(t, 2H, CH2-Ar), 2.21(t, 2H, CH2-CO-NH-), 2.35 (t, 2H, CH2-CO-O), 2.65 (t, 2H, CH2-Ar), 1.92 (m, 4H, C-CH2-C). 2.5. Synthesis of Fe3O4-DA-N3 NPs. Fe3O4 NPs (20 mg) was added into acetonitrile solution of dopamine-N3 1 (0.054 g/mL, 1 mL) in sample tube. The mixture solution was sonicated for 40 min, then


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the dopamine modified Fe3O4 NPs were separated through high speed centrifuges at 18.6g, washed four times with ethanol under an external magnetic field, then dried at 50 °C overnight. 2.6.

Preparation

propargyloxyethoxy)

of

alkyne-terminated

ethoxy]ethoxy}

maltose

ethanol-2,

3,

(2).

Into

6-tri-O-(2,

a 3,

solution 4,

of

2-{2-[2-(2-

6-tetra-O-acetyl)-β-D-

glucopyranosyl)-β-D-glucopyranoside (3.50g, 4.34 mmol)55,56 in dry MeOH (20 mL) was added 0.84 M NaOMe in MeOH (2.00 mL) at 0 ℃ under nitrogen atmosphere. The resulting mixture was allowed to stir at room temperature 2 h. The reaction mixture was neutralized by addition of Amberlite IR120 H until pH 6 and filtered off the resin. The filtrate was concentrated and dried under vacuum to give the product as a white solid. 1

H NMR (300 M Hz, CDCl3, δ, ppm) 5.37 (s, 2H), 4.23 (m, 3H), 3.94- 3.55 (m, 26H), 3.42- 3.37 (M,

1H), 2.91(s, 1H). HR MS (ESI) m/z Calcd for C23H40O15 [M+NH4]+: 574.2705, found 574.2716. 2.7. Preparation of Fe3O4-DA-Maltose NPs. Fe3O4-DA-N3 NPs (30 mg) and alkyne-terminated maltose (15 mg) were dispersed in MeOH/H2O (v/v, 1/1, 1.2 mL), then CuSO4·5H2O (12 mg) and (+)sodium L-ascorbate were (5 mg) were added in sequence. The reaction mixture was vibrated with an orbital shaker at room temperature for 24 h.36 The Fe3O4-DA-Maltose NPs were washed with deionized water and ethanol thrice, then dried at 50 °C for 3h. 2.8. Digestion of proteins. HRP was dissolved in NH4HCO3 (50 mM, pH 8.3) to the final concentration (1 mg/mL) 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) and incubated at 37 °C for 16 h. IgG was dissolved in NH4HCO3 (50 mM, pH 8.3) to the final concentration and denatured at 100 °C for 15 min. After that, the samples were reduced with 3.1 g DTT at 60 °C for 1 h and alkylated by 7.2 mg IAA at room temperature in the dark for 40 min. Then, trypsin was added into the solution at an enzyme/substrate ratio of 1:20 (w/w) and incubated at 37 °C for 16 h. The digestion of BSA was performed by incubating a mixture of 1 mg BSA, 100 µL 8 M urea and 20 µL in 50 mM NH4HCO3 solution (pH 8.3) at 56 °C for ACS Paragon Plus Environment

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1 h, and then mixing 20 µL 200 mM IAA at 37 °C in the dark for 30 min, then adding 860 µL NH4HCO3 solution and trypsin in the mixture at 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. 1 mg protein extraction from human renal mesangial cells was precipitation by TCA, the pellet was resuspended in 100 mM NH4HCO3. The sample was further reduced in 5 mM DTT, alkylated with 15 mM iodoacetamide. Trypsin was added with an enzyme/sample ratio of 1:50 for digestion overnight at 37°C. The resulting peptides were desalted and enriched using Sep-pak C18 cartridges (Waters Ltd, Elstree, UK), then evaporated to dryness. 2.9. Glycopeptides enrichment. Fe3O4-DA-Maltose NPs (or Fe3O4, Fe3O4-DA-N3 NPs, 1 mg) was diluted with loading buffer (80% ACN/H2O, 0.1 % FA) containing a determined amount of HRP digests. After incubation at room temperature for 1 h, it was then washed thirce with the 400 µL loading buffer with aid of an external field. Finally adsorbed glycopeptides were eluted from the composite with 10 µL elution buffer (10 % ACN/H2O, 1% FA) at room temperature for 30 min. The elutes were analyzed by MALDI-TOF MS. Fe3O4-DA-Maltose NPs (or Fe3O4, Fe3O4-DA-N3 NPs, 1 mg) was diluted with NH4HCO3 buffer (75% ACN/H2O, 25 mM) containing a determined amount of IgG digests. After incubation at room temperature for 1 h,, it was then washed thrice with 400 µL loading buffer. Then adsorbed glycopeptides were eluted from the composite with 10 µL NH4HCO3 buffer (20% ACN/H2O, 25 mM) at room temperature for 30 min. The elutes were analyzed by MALDI-TOF MS. For the glycopeptide enrichment from human renal mesangial cells, 400 µg of the digests was dissovled in 6 mL loading buffer (80% ACN/H2O, 0.1 % FA), incubated with 20 mg of Fe3O4-DAMaltose MNPs for 1 h, and subsequently washed thrice with 2 mL of loading buffer. Then, the trapped glycopeptides were eluted twice 400 µL of eluting buffer for 30 min, and the elution was evaporated to ACS Paragon Plus Environment

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dryness. The obtained glycopeptides were redissolved in 10 mM NH4HCO3, and 1000 unites of PNGase F was added. After the mixture was incubated at 37 °C for 18 h to remove the glycan moieties. Then the reaction was terminated by heating to 100 °C for 10 min, and the mixture was desalted and enriched using Sep-pak C18 cartridges (Waters Ltd, Elstree, UK) and evaporated to dryness, and redissolved prior to analysis by nano LC-MS/MS. 3. Results and Discussion 3.1. Preparation and characterization of Fe3O4-DA-Maltose NPs The size and morphology of the Fe3O4 and Fe3O4-DA-Maltose NPs were characterized by TEM. As shown in Fig. 1a, the diameter of resulting Fe3O4 NPs was 10∼20 nm. Due to the azido-terminated dopamine derivative formed the thinner layer on the surface of Fe3O4 through the coordination, after the hydrophilic maltose connecting dopamine did not result in greatly size change, and the modification layer of Fe3O4-DA-Maltose NPs was not detected (Fig. 1b). Furthermore, the Fe3O4, Fe3O4-DA-N3, Fe3O4-DA-Maltose NPs were characterized by Fourier transform infrared (FT-IR) spectroscopy (Fig. 2). In the spectrum of Fe3O4 NPs (Fig. 2a), the peak at 587 cm-1 was ascribed to the stretching vibration of Fe-O bond, and the broad peak centered at 3328 cm-1 was assigned to the N-H and/or O-H vibration on the surface of Fe3O4 NPs. Compared with the above spectrum, the additional peaks appeared in the FTIR spectrum of Fe3O4-DA-N3 NPs (Fig. 2b). The peak at 2104 cm-1 was assigned to the stretching vibration of azido groups of dopamine-N3, and the characteristic absorption peaks of benzene ring moiety located at 1639∼1484 cm-1. The peaks at 1266 and 1734 cm-1 were ascribed to the stretching vibration of C-O and C=O bond of ester group, respectively. In the spectrum of Fe3O4-DA-Maltose NPs (Fig. 2c), the broad peak at 1624 cm-1 was attributed to the superposition of characteristic absorption peaks of benzene ring and triazole. The appearance of peak at 1079 cm-1 was assigned to C-O-C bond of alkyl chain. Meanwhile, the complete disappearance of the azide group typical adsorption peak indicated that the N3 group on the surface of MNPs reacted efficiently with triple bond terminated maltose by click chemistry. ACS Paragon Plus Environment

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The XRD patterns of the Fe3O4, Fe3O4-DA-N3 and Fe3O4-DA-Maltose NPs are shown in Fig. S1 (Supporting Information). In the 2θ range of 3-80°, the characteristic peaks for Fe3O4 (2θ=29.9°, 35.4°, 43.0°, 53.3°, 57.0°, 62.6°) at the corresponding 2θ values are indexed as (220), (311), (400), (422), (511) and (440), respectively, which can be indexed to the face center-cubic phase of Fe3O4 (JCPDS Card no. 19-629). XRD results confirmed that nanocrystalline structure remains unchanged before and after modification of maltose. The magnetic properties of the synthesized MNPs were studied using a vibrating sample magnetometer (VSM) at room temperature. As shown in Fig. 3, the saturation magnetic (Ms) values of Fe3O4, Fe3O4DA-N3 and Fe3O4-DA-Maltose NPs were 61.00, 58.84, 54.27 emu·g-1, respectively. With each step modification, the magnetization values of resulting NPs gradually decreased. Nevertheless, Fe3O4-DAMaltose NPs possess great Ms which show a fast response to the applied magnetic field (2000 Oe). After dispersing the Fe3O4-DA-Maltose NPs in water by shaking, they can be easily separated within twenty seconds with the an external magnetic field (Fig. 3 inset). This suggests that the novel hydrophilic MNPs possess excellent magnetic and redispersibility, which is advantageous to the application. XPS was further used to characterize the surface composition of the three kinds of MNPs (Fe3O4, Fe3O4-DA-N3 and Fe3O4-DA-Maltose) (Fig. 4). The XPS spectrum of Fe3O4-DA-N3 NPs showed a C1s peak around 284.0 eV, O1s peak at 530.2 eV and Fe signals at about 55.8 eV for Fe3p, 710.3 and 724.1 eV for Fe2p. Meanwhile, the N3 group is revealed by the bands at 404.5 (N=N+=N-) and 400.7eV (N=N+=N-), suggesting the presence of dopamine-N3 on the MNPs surface (Fig. 4 curve b). The disappearance of the signal of the N1s peak in XPS spectrum (Fig. 4 curve c) proved the successful binding of Fe3O4-DA-N3 to alkyne-terminated maltose. Meanwhile, water contact angle is employed to evaluate the relative surface hydrophilicity of Fe3O4DA-N3 NPs and Fe3O4-DA-Maltose NPs. To chatacterizate the surface hydrophilicity of the novel NPs, water contact angle was measured with powder tabletting method (Fig. 5). Deionized water was used as ACS Paragon Plus Environment

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a probe liquid and when de-ionized water was put on the membrane surface, the water contact angle was immediately averaged at five points. Generally, the smaller contact angle indicates the better hydrophilicity of materials. The contact angle of Fe3O4, Fe3O4-DA-N3, Fe3O4-DA-Maltose NPs were 22.6°, 33.4° and 17.1° respectively, which implying that the modification of maltose is helpful to enhancing the hydrophilicity of NPs. 3.2. Selective enrichment of glycopeptides The selective recognition of Fe3O4-DA-Maltose NPs to glycopeptides was based on the hydrophilic interactions between maltose and glycan moieties. To evaluated the selectivity and sensitivity of the target nanoparticles towards glycopeptides, N-glycosylated peptides of HRP was employed as test sample. The direct analysis of HRP digest (10 ng µL-1) without enrichment, only five glycosylated peptides labeled with H1, H4, H5, H7, H8, respectively (Table S1, see Supporting Information) could be observed, due to the suppression of highly abundant non-glycopeptides (Fig. 6A). And the signal peak is very weak. After enrichment with Fe3O4-DA-Maltose NPs, the eight glycosylated peptides were identified, meanwhile, the most abundant non-glycopeptides were efficiently removed (Fig. 6B). The surface decoration is based on the hydrophilic magnetic oxide iron. The improvement in hydrophilicity after assembly of maltose on the surface of Fe3O4 NPs was demonstrated by the comparsion of the Fe3O4 and Fe3O4-DA-N3 NPs used to enrich glycopeptides from HRP digests (Fig. 6C, D). The signals of glycosylated peptides were detected, however, non-glycosylated signals were identified and the signal strength is higher than glycopeptides. This indicates that the modification of maltose on the surface of Fe3O4 NPs contributed to the improvement of its hydrophilicity, which was consistent with the results of contact angle measurement. Meanwhile, the theoretical glycosylation sites of HRP, occupied by heterogeneous high-mannose type oligosaccharides, were also identified in the Table S1 (Supporting Information). The sensitivity of the Fe3O4-DA-Maltose NPs was detected with different concentrations of the tryptic HRP digest (Fig. 7). The signal intensity of glycopeptides gradually weakened by decreases of ACS Paragon Plus Environment

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concentration of HRP digests. When the concentration of HRP digests was 0.05 ng µL-1 (Fig. 7D), two glycopeptide peaks was still observed after enrichment with Fe3O4-DA-Maltose from HRP digests. To evaluate the discrimination performance between non-glycopeptides and glycopeptides, the mixture of HRP and BSA tryptic digests at a mass ratio of 1:1 and 1:10 was enriched by Fe3O4-DAMaltose NPs. As shown in Fig. 8, most detected glycopepitdes in the pure HRP peptide sample also appeared. In order to further demonstrate the universality of this composite for selective enrichment of glycopeptides from the biological sample, Fe3O4-DA-Maltose NPs were applied to isolate/enrich IgG tryptic digests. The different loading buffer was used for glycopeptides enrichment from HRP and IgG digests. Because the surface properties of the Fe3O4-DA-Maltose NPs affect the enrichment of glycopeptides, especially for the complex biological samples, the zeta potential value of Fe3O4-DAMaltose NPs under different pH was investigated. As shown in Fig. S2 (Supporting Information), the zeta potential value of Fe3O4-DA-Maltose NPs is 0.40 mV at acidic loading buffer (pH 3.0, 80% ACN/H2O, 0.1% FA), which exhibited the Fe3O4-DA-Maltose NPs are positively charged under acidic condition. The Fe3O4-DA-Maltose NPs still have electrostatic interactions to sialic acid-terminated glycopeptides which exist in IgG digests. The zeta potential value of Fe3O4-DA-Maltose NPs decreased to -17 mV under pH 9.20, this could suppress the electrostatic interaction between Fe3O4-DA-Maltose NPs and sialic acid-terminated glycopeptides. The basic buffer (pH 9.20, 75 % ACN/25 mM NH4HCO3) was used as loading buffer in glycopeptides enrichment from IgG digest. The direct analysis of IgG tryptic digest without enrichment showed only 2 weak peaks belonging to glycopeptides labelled with I1 and I6 in the Table S2 (Supporting Information) due to the suppression of many dominant peaks of abundant non-glycopeptides existed in the mixture. After enrichment with Fe3O4-DA-Maltose NPs (Fig. 9B), the additional 15 glycopeptides were identified except for peak I1 and I6. Moreover, the most nonglycosylated peptides existing in IgG tryptic digests was removed efficiently, and the signal-to-noise (S/N) of the glycopeptides was enhanced obviously. In comparsion with the enrichment efficiency of Fe3O4-DA-Maltose NPs to IgG digests, we also applied Fe3O4 and Fe3O4-DA-N3 NPs to isolate/enrich ACS Paragon Plus Environment

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IgG tryptic digests, respectively. As shown in Fig. 9C and 9D, the signal of glycopeptides were completely suppressed. Meanwhile, only 4 weak peaks of glycopeptides were detected. To further evaluate their ability to enrich glycopeptides in the complex samples, the Fe3O4-DAMaltose NPs were applied to identify the N-linked glycopeptides from the human renal mesangial cells (HRMC). HRMC serve as a filtration barrier of the kidney. The injury of mesangial cells could cause diabetic nephropathy, leading end-stage renal disease. Emerging evidence indicates that mesangial cells can be damaged by high glucose, however the mechanism is unclear. Herein we used the novel materials to enrich the N-link glycopeptide from the HRMC (cell culture and protein extraction, see Supporting Information) which was treated with high glucose. The glycopeptide enrichment was further analyzed by HPLC-MS/MS (Supporting Information). Finally, 115 different N-linked glycopeptides were identified, representing 93 gene products and 124 glycosylation sites in HRMC (Table S3, Supporting Information). 3.3. Evaluation of binding capacity and recovery of Fe3O4-DA-Maltose NPs for glycopeptide enrichment Different amounts (5-100 µg) of Fe3O4-DA-Maltose NPs were used to treat 3 µg human IgG digest. After the enrichment, the elution (1 µL from 20 µL total) was analyzed with MALDI-TOF MS. When the peak intensity of six selected glycopeptides reach maximum, the total amount of glycopeptides were bonded onto the NPs. The binding capacity was calculated by the amount of human IgG (3 µg) to NPs. As shown in Fig. S3 (Supporting Information), the N-glycopeptides within 3 µg IgG digest can be almost captured by 70 µg of Fe3O4-DA-Maltose NPs and the binding capacity of MNPs was about 43 mg g-1. Stable-isotope dimethyl labeling was used to study the recovery yield of Fe3O4-DA-Maltose NPs for glycopeptides enrichment according to a previous report.57 The recovery was calculated by the peak intensity ratio of the heavy-tagged glycopeptides to light-tagged glycopeptides. The recovery of


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glycopeptides ranged from 85% to 110%, which confirmed that Fe3O4-DA-Maltose NPs possess great potential for the enrichment of glycopeptides. 4. Conclusions In summary, an novel hydrophilic maltose-coated Fe3O4 magnetic nanoparticles (Fe3O4-DA-Maltose NPs) were synthesized successfully with simple synthetic route and convenient operation “click chemistry” and “metal chelation”. Moreover, compared with precursor, the hydrophilicity of maltosefunctionalized MNPs was distinctly improved, which was demonstrated by enrichment efficiency of glycopeptides. The resulting Fe3O4-DA-Maltose NPs exhibited high selectivity and detection sensitivity for the enrichment of glycopeptides from tryptic HRP, IgG digests and the real biological sample (human renal mesangial cells). Consequently, we anticipate that biocompatible dopamine derivative assembled onto iron oxide MNPs can be a powerful tool for identification of protein glycosylation in various biological samples. Supporting Information Detail procedure of cell culture and protein extraction, LC-MS/MS analysis and MS/MS data analysis; XRD patterns of Fe3O4 , Fe3O4-DA-N3, and Fe3O4-DA-Maltose NPs; zeta spectra of Fe3O4-DA-Maltose NPs on the change of pH; the amount of Fe3O4-DA-Maltose NPs influencing intensity of six selected glycopeptides from tryptic digests of human IgG; identified glycopeptides and glycan structures of human HRP, IgG digests and human renal mesangial cells after enrichment by Fe3O4-DA-Maltose NPs. Acknowledgements The authors are grateful to the National Basic Research Program of China (No. 2012CB910601), the National Natural Science Foundation of China (No. 21275080, 21475067), the Research Fund for the Doctoral Program of Higher Education of China (No. 20120031110007) and the Natural Science Foundation of Tianjin (No. 15JCYBJC20600).


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Figure Captions: Scheme 1 Schematic illustration of fabrication of Fe3O4-DA-Maltose NPs, selective enrichment strategy and MS analysis of glycopeptides. Figure 1 TEM images of Fe3O4 (a) and Fe3O4-DA-Maltose (b) NPs. Figure 2 FT-IR spectra of Fe3O4 (a), Fe3O4-DA-N3 (b) and Fe3O4-DA-Maltose (c) NPs. Figure 3 Magnetic hysteresis curves of Fe3O4 (a), Fe3O4-DA-N3 (b) and Fe3O4-DA-Maltose (c) NPs. Figure 4 X-ray photoelectron spectra (XPS) of Fe3O4 (a), Fe3O4-DA-N3 (b) and Fe3O4-DA-Maltose (c) NPs. Figure 5 Shape of water drops on Fe3O4 (A), Fe3O4-DA-N3 (B), Fe3O4-DA-Maltose (C) NPs. Figure 6 MALDI-TOF-MS analysis of tryptic digests of HRP (10 ng µL-1): (A) direct analysis; after enrichment with (B) Fe3O4-DA-Maltose, (C) Fe3O4 and (D) Fe3O4-DA-N3 NPs. The peaks of glycopeptides are marked with H. Figure 7 MALDI-TOF-MS analysis of different concentration of HRP tryptic digests: (A) 1, (B) 0.5, (c) 0.2, (D) 0.05 ng µL-1 HRP after enrichment with Fe3O4-DA-Maltose NPs. The peaks of glycopeptides are marked with H. Figure 8 MALDI-TOF-MS analysis of tryptic BSA and HRP after enrichment. The mass ratio of BSA:HRP are 1:1 (A), 10:1 (B). The peaks of glycopeptides are marked with H. Figure 9 MALDI-TOF-MS analysis of tryptic digests of IgG (10 ng µL-1): (A) direct analysis; after enrichment with (B) Fe3O4-DA-Maltose, (C) Fe3O4 and (D) Fe3O4-DA-N3 NPs. The peaks of glycopeptides are marked with I.


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Scheme 1


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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 6


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Figure 8


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Graphic for manuscript 165x76mm (300 x 300 DPI)


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Click Synthesis of Hydrophilic Maltose-Functionalized Iron Oxide

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