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Click synthesis of glucose-functionalized hydrophilic magnetic mesoporous nanoparticles for highly selective enrichment of glycopeptides and glycans
Accepted Manuscript Title: Click synthesis of glucose-functionalized hydrophilic magnetic mesoporous nanoparticles for highly selective enrichment of glycopeptides and glycans Author: Jiangnan Zheng Yun Xiao Ling Wang Zian Lin Huanghao Yang Lan Zhang Guonan Chen PII: DOI: Reference:
S0021-9673(14)01013-9 http://dx.doi.org/doi:10.1016/j.chroma.2014.06.070 CHROMA 355549
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
18-12-2013 11-6-2014 22-6-2014
Please cite this article as: J. Zheng, Y. Xiao, L. Wang, Z. [email protected], H. Yang, L. [email protected], G. Chen, Click synthesis of glucosefunctionalized hydrophilic magnetic mesoporous nanoparticles for highly selective enrichment of glycopeptides and glycans, Journal of Chromatography A (2014), http://dx.doi.org/10.1016/j.chroma.2014.06.070 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Click synthesis of glucose-functionalized hydrophilic magnetic
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mesoporous nanoparticles for highly selective enrichment of
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glycopeptides and glycans
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Jiangnan Zheng, Yun Xiao, Ling Wang, Zian Lin,* Huanghao Yang, Lan Zhang,*
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and Guonan Chen
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Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian
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Provincial Key Laboratory of Analysis and Detection Technology for Food Safety,
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Department of Chemistry, Fuzhou University, Fuzhou, Fujian, 350108, China
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First corresponding author: Zian Lin;
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Second corresponding author: Lan Zhang
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Postal address: Department of Chemistry, Fuzhou University,
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Fuzhou, Fujian, 350108, China
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Fax: 86-591-22866135
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E-mail: [email protected] (Z.A. Lin); [email protected] (L.Zhang);
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Abstract 1
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Selective enrichment of glycopeptides from complex biological samples is essential for
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MS-based glycoproteomics, but challenges still remain. In this work, glucose-
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functionalized magnetic mesoporous nanoparticles (MMNs), which hold the attractive
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features of well-defined core/shell structure, high specific surface area (324 m2/g), narrow
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pore size distribution (2.2 nm) and high magnetic responsivity (69.1 emu/g), were
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synthesized via click chemistry and applied to enrich glycopeptides and glycans. Taking
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advantages of the size-exclusive effect of mesopore against proteins and the hydrophilic
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interaction between glycans and glucose, the hydrophilic MMNs possessed high
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selectivity for glycopeptides at the digested mixtures of horseradish peroxidase (HRP),
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myoglobin and β-casein at molar ratio of 1:1:10, large enrichment capacity (as high as
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250 mg/g), high sensitivity (50 fmol), excellent speed (5 min for enrichment) and high
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recovery of glycopeptides (as high as 94.6%). Additionally, the MMNs exhibited
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excellent performance in enrichment of N-linked glycans from the digested human serum
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that are made up of peptides, large proteins and other compounds. These outstanding
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features will give the hydrophilic MMNs high benefit for MS analysis of low-abundance
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glycopeptides/glycans.
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Keywords:
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glycopeptide, glycan, enrichment
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magnetic
mesoporous
nanoparticles,
click
chemistry,
hydrophilic,
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1. Introduction
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Protein glycosylation, as one of the most ubiquitous and complex post-translational
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modifications, plays a vital role in many biological processes [1,2]. Numerous studies
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have revealed that many clinical biomarkers are glycoproteins. Currently, mass
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spectrometry (MS)-based technique has been a preferred choice in glycoproteome
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analysis. However, owing to the inherent low abundance and poor ionization efficiency
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of glycopeptides, enrichment and identification of glycopeptides is still an enduring
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challenge. In the past few years, considerable efforts have been devoted to developing
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methods for isolation and enrichment of glycopeptides including lectin-based affinity
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chromatography [3], boronate affinity chromatography [4], hydrazine chemistry [5],
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titanium dioxide [6], and hydrophilic interaction chromatography (HILIC) [7-9]. Among
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these methods, HILIC have attracted wide interest because of its good reproducibility,
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broad glycan-binding specificity, and excellent compatibility with MS analysis.
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Several hydrophilic absorbents/stationary phases have been widely used in recent years
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for glycoprotein/glycopeptide enrichment, including sepharose [10], cellulose [11],
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zwitterionic monomers [12], and maltose [13, 14]. Recently, Xiong et al. prepared
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branched PEG brushes hybrid magnetic nanoparticles (MNPs) and then the reactive
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hydroxyl groups were modified with maltose, which could increase the amount of
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immobilized maltose and provide high selectivity and sensitivity for glycopeptide
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detection[8]. However, the synthesis processes were tedious. More recently, Xiong et al.
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fabricated multilayer polysaccharide shells coated MNPs using a layer-by-layer (LBL)
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approach via the alternate deposition of hyaluronan (HA) and chitosan (CS) onto the
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surface, and the MNPs-(HA/CS)10 exhibited high selectivity, high detection sensitivity,
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large binding capacity for the enrichment of glycopeptides[9]. Yet, the enrichment
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recovery of MNPs (69%) needs to be improved. And the LbL-assembled nanostructure
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may suffer from a lack of stability when they are exposed to certain changes in aqueous
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environments [15]. Therefore, developing an effective and robust HILIC method for
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glycopeptide enrichment is highly desirable.
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Ordered mesoporous materials have attracted immense interest in the fields of
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nanosensor, catalyst, and biomedical engineering due to the large surface area, well-
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defined mesoporous structure, and narrow pore size distribution [16]. The application of
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mesoporous materials in biological sample pretreatment, such as selective capture of
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endogenous peptides [17], phosphopeptides [18] and glycans [19,20], has been reported
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recently, where the unique mesoporous structures were superior in capturing target
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peptides and repelling against interfering large molecular weight proteins due to the size-
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exclusion effect. Meanwhile, magnetic materials have received great attention due to their
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magnetism properties, and have been applied extensively in proteomics, including protein
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digestion and proteins/peptides enrichment [21-25]. More recently, taking advantages of
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magnetic materials and mesoporous structure, Lu et al. [26] developed a novel method to
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synthesize magnetic mesoporous Fe3O4@mTiO2 microspheres with high surface area,
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large pore volume, and high magnetic susceptibility, which possessed remarkable
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selectivity and high enrichment capacity for phosphopeptides. Nevertheless, no reports on
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hydrophilic magnetic mesoporous materials for selective isolation and enrichment of
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glycopeptides and glycans have been found so far.
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Herein, we report a facile approach for the synthesis of glucose-functionalized
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hydrophilic magnetic mesoporous nanoparticles (MMNs) via click reaction and applied
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them for selective isolation and enrichment of glycopeptides/glycans. The hydrophilic
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MMNs hold the features of high surface area and glycopeptide-suitable pore size, and
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thus can be employed to enrich glycopeptides/glycans from complex biological samples.
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Besides, with the unique magnetic responsivity, they can be easily removed from the
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mixture by employing an external magnet, rather than with the help of centrifugation.
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This proposed approach offered higher efficiency in enrichment of glycopeptides/glycans
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than the previous reported methods.
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2. Experimental
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2.1. Chemicals and reagents
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Sodium citrate dehydrate (Na3Cit·2H2O), ethylene glycol (EG), and copper (II) sulphate
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pentahydrate (CuSO4·5H2O) were obtained from Sinopharm Chemical Reagent, Co., Ltd
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(Shanghai, China). Iron (III) chloride (FeCl3), sodium ascorbate, tetraethylorthosilicate
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(TEOS), 3-chloropropyltriethoxysilane (CPTES), D-glucose, zinc dust (99.99%),
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cetyltrimethylammonium bromide (CTAB), formic acid (FA) and sequencing-grade
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modified trypsin (TPCK-trypsin) were obtained from Aladdin Chemistry Co., Ltd
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(Shanghai, China). Propargyl bromide (80 wt% solution in toluene) was purchased from
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J&K Chemical Ltd (Shanghai, China). HRP was purchased from Shanghai Lanji Co. Ltd.
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(Shanghai, China). Sodium azide (NaN3), horse myoglobin (Mb, Sigma cat. no. M1882),
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and β-casein from bovine milk (Sigma C6905) were the products of Sigma-Aldrich (St.
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Louis, USA). Peptide N-glycosidase (PNGase F) was purchased from New England
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Biolabs (Ipswi ch, MA, USA). Centrifugal filter with MWCO of 10 kDa was purchased
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from Millipore (Bedford, MA). Deionized water was prepared with a Milli-Q water
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purification system (Millipore, Milford, MA). All other chemicals and reagents were of
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analytical grade or better.
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2.2. Preparation of Fe3O4 magnetic nanoparticles The magnetic nanoparticles (MNPs) were prepared according to the previous
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description [27] with some modifications. Briefly, FeCl3 (6.8 g), sodium acetate (12.0 g),
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Na3Cit·2H2O (2.0 g) were dissolved in EG (200 mL). The obtained homogeneous yellow
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solution was transferred to autoclave, and then heated to 200 °C for 7 h. After reaction,
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the product was collected with a magnet and washed with water and ethanol for several
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times and then dried at room temperature.
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2.3. Preparation of Fe3O4@mSiO2-Cl MMNs
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The synthesized Fe3O4 MNPs were coated with mesoporous silica shells by hydrolysis
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and condensation of TEOS and CPTES. Typically, the Fe3O4 nanoparticles (0.5 g) and
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CTAB (1.0 g) were added into 500 mL H2O, and ultrasonically dispersed for 1 h to form
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a stable dispersion. Afterward, 80 mL of NaOH aqueous solution (7.5 mM) was added to
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above mixture and further stirred at 60 °C for another 30 min. Then, 20 mL of
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TEOS/CPTES/ethanol (v/v/v: 2/1/2) solution was added by injection under mechanical
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stirring, and subsequently the dispersion was further heated at 60 °C for 12 h. The
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obtained product (Fe3O4@SiO2) was collected by magnetic separation and washed with
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ethanol repeatedly, and redispersed in ethanol (60 mL) for refluxing at 90 °C to remove
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the CTAB. The refluxing process was repeated 5 times to achieve a complete removal of
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CTAB, and the resulting Fe3O4@mSiO2–Cl MMNs were dried at 50 °C for 24 h for the
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future use.
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2.4. Preparation of Fe3O4@mSiO2-N3 MMNs
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Fe3O4@mSiO2-Cl MMNs (0.5 g) were dispersed in 100 mL saturated solution of NaN3
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in DMF. The obtained mixture was stirred at 80°C for 24 h. After reaction, the obtained
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product (Fe3O4@mSiO2-N3) was collected by magnetic separation, washed with ethanol
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repeatedly, and dried at room temperature.
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2.5. Synthesis of propargyl glucose Propargyl glucose was obtained through direct addition of propargyl bromide to
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aldehyde substrates mediated with low valent iron [28]. Briefly, a mixture of D-glucose
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(0.90 g, 5 mmol), propargyl bromide (1.6 mL, 80 wt% solution in toluene, 15 mmol), and
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FeCl3 (2.43 g, 15 mmol) in THF (100 mL) was stirred thoroughly in a water bath at
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around 10~15 °C (maintained by addition of pieces of ice). After stirring the mixture for
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10 min, zinc dust (0.975 g, 15 mmol) was added portion wise over a period of 8 min. The
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mixture was stirred at room temperature for 12h. After reaction, the mixture was then
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treated successively with diethyl ether (100 mL) and water (50 mL), stirred for 10 min
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and then filtered. The filtrate was treated with ammonium bicarbonate to precipitate metal
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ions and then filtered. The aqueous layer was used without drying.
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2.6. Preparation of Fe3O4@mSiO2-Glucose MMNs
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Fe3O4@mSiO2-N3 MMNs (0.5 g) was dispersed in 100 mL H2O. Then, the propargyl
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glucose solution (in 100 mL water) was added to the above mixture, followed by
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CuSO4·5H2O (372 mg, 1.5 mmol) and sodium ascorbate (892 mg, 4.5 mmol). The
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reaction mixture stirred in air at RT for 12 h. After reaction, the product was washed with
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water and ethanol for several times and dried at room temperature.
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2.7. Characterization
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Scanning electron microscopy (SEM) images were obtained with FEI Inspect F50 (FEI,
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USA). Transmission electron microscopy (TEM) analysis was performed on a FEI Tecnai
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G2 20 (FEI, USA) at 200 kV. Nitrogen adsorption and desorption isotherms were
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measured using an ASAP 2020 (Micromeritics, USA). The samples were degassed in a
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vacuum at 200 °C for 8 h prior to measurement. Brunauer–Emmett–Teller (BET) method
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was utilized to calculate the specific surface areas (SBET) using adsorption data in a
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relative pressure range from 0.18 to 0.35. By using the Barrett-Joyner-Halenda (BJH)
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model, the pore volume and pore size distribution were derived from the adsorption
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branches of the isotherms, and the total pore volumes were estimated from the adsorbed
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amount at a relative pressure P/P0 of 0.992. Fourier-transform infrared (FT-IR) spectra
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were taken on Nicolet 6700 spectrometer (Thermo Fisher, USA) using KBr pellets. The
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crystal structure of the MMNs was determined by X'Pert-Pro MPD (Philips, Holland).
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The magnetization curves were carried out at room temperature on the superconducting
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quantum interference device magnetometer (SQUID) MPMS XL-7 (Quantum Design,
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San Diego, USA).
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2.8. Release of N-Linked glycans from human serum
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To release the N-linked glycans, human serum were thawed at 4 ºC and centrifuged at
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13000g for 10 min. The collected supernatants (50 μL) were diluted to 500 μL by 10 mM
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ammonium bicarbonate (pH 7.5). Then the solutions (100 μL) were boiled for 5 min,
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cooled to room temperature, and incubated at 37 ºC for 24 h after adding 10 U of PNGase
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F.
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2.9. Enrichment of N-Linked glycopeptides and glycans
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Tryptic digests of protein were dissolved in 200 μL binding buffer (80% ACN
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containing 0.2% FA). Then, 60 μg Fe3O4@mSiO2–Glucose MMNs were suspended in
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above solution in a microcentrifuge tube. After incubated for 5 min, the MMNs having
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captured the glycopeptides were isolated from the solution with an assistant magnet and
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washed three times with binding buffer in order to remove residual uncaptured peptides.
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Finally, the captured peptides were eluted with 10 μL elution buffer (20% ACN
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containing 0.2% FA) for 5 min, and the eluate was analyzed by ESI-Q-TOF MS. To enrich the N-linked glycans, the diluted serum digestion (2.5 μL) was dissolved in
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400 μL binding buffer (80% ACN containing 0.2% FA). Then, the Fe3O4@mSiO2–
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Glucose MMNs (200 μg) were suspended in the solution to enrich glycans. The other
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procedures for the enrichment of N-linked glycans from human serum were the same as
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for the above mentioned glycopeptides.
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2.10. Mass spectrometry
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A 1200 series HPLC system with a binary SL pump was used. Detection was
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performed using an Agilent 6520 Q-TOF with dual electrospray ion source (ESI). The
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separation of all samples was performed on Agilent Poroshell 120 EC-C18 column
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(2.7 μm, 3.0 mm × 50 mm, Agilent). The flow rate was 0.3 mL/min. Solvent A was
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composed of water containing 0.1% formic acid. Solvent B was composed of ACN
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containing 0.1% formic acid. The following gradient program was used: 0–3 min, 3% B;
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3–20 min, 3–40% B; 20–23 min, 40–80% B; 23–25 min, 80% B. The sample injection
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volume was 3 μL. Nitrogen was used as drying gas at a temperature of 350°C and a flow–
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rate of 10 L/min. Full scan MS data and MS/MS data were acquired at m/z 800-2000 and
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100-3000, respectively. Scan rates for MS and MS/MS data were set to 3 spectra/sec. The
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voltage set for the MS capillary was 4 kV and the fragmentor was set to 175 V. For MS2
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experiments, the collision energy was set to according to formula (see supporting
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information). GlycoWorkbench [29] software was applied for mass spectrometric data
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interpretation and glycoform analysis.
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3. Results and discussion
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3.1. Synthesis and characterization of hydrophilic MMNs The general scheme for the synthesis of hydrophilic MMNs is illustrated in Fig. 1.
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Firstly, using CTAB as a structural directing agent, TEOS and CPTES as the precursors,
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a CTAB/mSiO2–Cl composite was deposited on the Fe3O4 MNPs by sol-gel process.
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Secondly, CTAB templates were removed by ethanol extraction to form a mesoporous
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SiO2 shell, resulting in well-dispersed Fe3O4@mSiO2–Cl MMNs. Thirdly, the
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chloropropylated MMNs were treated with NaN3 to obtain Fe3O4@mSiO2–N3 MMNs.
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Finally, Fe3O4@mSiO2–Glucose MMNs were synthesized by click reaction between
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Fe3O4@mSiO2–N3 MMNs and propargyl glucose.
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Representative SEM and TEM images showed that the synthesized Fe3O4@mSiO2–
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Glucose MMNs were approximately spherical in shape with a core-shell structure, which
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consisted of a magnetic core (ca. 200 nm) and a thin mesoporous SiO2 shell (ca. 15 nm)
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with perpendicularly oriented channels (Fig. 2A and B). The short channels of
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Fe3O4@mSiO2–Glucose MMNs might improve the mass transfer efficiency during
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enrichment of low molecular weight (MW) biomolecules [20]. The magnetic core with
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narrow size distribution was obtained by means of a solvothermal reaction, and could
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well disperse in aqueous solution (Fig. 2C). After sol-gel process, the color of the MMNs
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changed from black to bright orange (Fig. 2D), indicating the successful coating of silica
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shell as well. The functionalized MMNs also exhibited high dispersibility in aqueous
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phase.
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The structural properties of the Fe3O4@mSiO2–Glucose MMNs were studied by
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nitrogen adsorption-desorption at 77 K, as shown in Fig. 3A. The Fe3O4@mSiO2–
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Glucose MMNs exhibited a type IV isotherm, which was typical for mesoporous silica
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synthesized with CTAB as surfactant. From pore size distribution curve, it was observed
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that the pore diameter of the MMNs was about 2.2 nm and displayed a narrow size
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distribution. The BET surface area and total pore volume were calculated to be 324 m2/g
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and 0.25 cm3/g, respectively. Taking together, these results showed that the core-shell
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structure of Fe3O4@mSiO2–Glucose MMNs had a large specific surface area and large
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pore volume, which was caused by the existence of mesoporous silica coating.
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Significantly, the high degree of porosity and suitable mesopore size of these MMNs
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made it possible to selectively extract low MW biomolecules from complex samples.
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FT-IR spectra (Fig.3B) provided a direct proof for synthetic process of the
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Fe3O4@mSiO2–Glucose MMNs. The spectrum of Fe3O4@mSiO2–Cl MMNs with CTAB
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templates was showed in curve a. The typical absorption bands at 584 and 1080 cm−1
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could be assigned to Fe–O–Fe and Si–O–Si vibrations, respectively. Moreover, the strong
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absorption peak at 2925 and 2850 cm−1 assigned to –CH2 groups indicated the successful
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coating of CTAB-templated silica layer. After refluxing process, the absorption peak of –
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CH2 became weak (curve b), confirming the removal of CATB. Compared to spectrum b,
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the appearance of a characteristic peak at 2104 cm−1 due to the out-of-phase stretching
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vibration of azido groups in spectrum of Fe3O4@mSiO2–N3 MMNs (curve c) clearly
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suggested that the chlorine atoms had been substituted by N3 groups. The spectrum of
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Fe3O4@mSiO2–Glucose MMNs (curve d) showed the disappearance of the N3 vibration
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band at 2104 cm−1, which indicated all N3 groups did react with propargyl glucose by
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virtue of a highly efficient alkyne-azide click reaction [30]. Meanwhile, the band
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intensity of –CH2 groups at 2945 and 2875 cm−1 was increased after click reaction, which
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also implied the successful surface modification of MMNs with glucose. The crystalline structures of Fe3O4 and Fe3O4@mSiO2–Glucose MMNs were
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determined by XRD (Fig. 3C). The positions and relative intensities of all diffraction
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peaks for Fe3O4 and Fe3O4@mSiO2–Glucose MMNs matched well with those from the
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JCPDS card (19-629) for magnetite. The broad diffraction peaks suggested the magnetite
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core was composed of small nanocrystals [27]. The size of these small nanocrystals,
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calculated from the Debye-Scherrer formula for the strongest (311) diffraction peak, was
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11.8 nm. The unique structure allowed MMNs to retain superparamagnetic behaviour at
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room temperature even though their size exceeded 30 nm. The magnetic characterization
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on a SQUID (Fig. 3D) confirmed that both of the bare Fe3O4 MNPs and Fe3O4@mSiO2–
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Glucose MMNs exhibited superparamagnetic behavior at 300K. No remanence remained
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when the applied magnetic field was removed. Their corresponding saturated
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magnetization values were 79.8 and 69.1 emu g−1, respectively. Such high saturation
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magnetization endued the MMNs with a fast response to an external magnetic field. (Fig.
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2D and E). It was suggested that the MMNs possessed excellent magnetic responsiveness.
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3.2. Selective enrichment of glycopeptides from HRP digest
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To evaluate the selectivity and enrichment efficiency of the Fe3O4@mSiO2–Glucose
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MMNs, tryptic HRP digest was employed as a model sample. It is well known that HRP
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has nine potential glycosylation sites of which at least eight sites are occupied by
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heterogeneous high-mannose type oligosaccharides [31]. It is generally acceptable that
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glycopeptides would be bound more strongly than nonglycopeptides on Fe3O4@mSiO2–
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Glucose MMNs based on the hydrophilic interactions between glucose and glycan
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moieties. In order to make the hydrophilic interaction play a dominant role for
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glycopeptide enrichment, ACN/H2O/FA (80/19.8/0.2, v/v/v) was recommended as
276
loading buffer. Fig. 4 showed the direct MS analysis of 0.5 ng/μL HRP tryptic digest treated with and
278
without Fe3O4@mSiO2–Glucose MMNs. Without treatment, only 4 peaks that matched
279
the glycopeptides (the identified N-glycopeptides was displayed in Table S1, see
280
supporting information) could be determined from 0.5 ng/μL tryptic HRP digestion (Fig.
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4A). In contrast, after treatment with the MMNs, up to 20 peaks corresponding to 18 N-
282
glycopeptides could be clearly detected with higher signal to noise ratios (S/N) (Fig. 4B).
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The oligosaccharide composition and amino sequence of these detected glycopeptides
284
(Table 1) was in agreement with the previous report describing a xylosylated, core-(α1-
285
3)-fucosylated trimannosyl N-glycan structure as the predominant species at all N-
286
glycosylation sites of HRP [31]. Furthermore, seven HRP peptides of unreported
287
structure were confirmed as glycopeptides according to their product ions at m/z
288
163.0601 (Man+), m/z 204.0866 (GlcNAc+), m/z 366.1394 (Man1GlcNAc1+), m/z
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528.1923
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(Man3GlcNAc1+) and 822.2873 (XylMan3GlcNAc1+), which were marker oxonium ions
291
of glycopeptides (Fig. S1, see supporting information). Furthermore, the sensitivity was
292
further evaluated by the enrichment of glycopeptides from low concentration of HRP
293
digest (0.011 ng/μL, corresponding to 50 fmol HRP), two glycopeptides were still
294
observed after enrichment (Fig. S2, see supporting information). The results indicated
295
that the Fe3O4@mSiO2–Glucose MMNs had predominant enrichment efficiency and high
296
selectivity for the glycopeptides. Totally 8 N-glycosylation sites (Table 1) of HRP were
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(Man2GlcNAc1+),
m/z
660.2345
13
(XylMan2GlcNAc1+),
m/z
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Page 13 of 31
identified by the hydrophilic MMNs, which exhibited better enrichment performance than
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PEG-Maltose hybrid MNPs (6 identified glycosylation sites of HRP) [8]. Moreover,
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owing to the unique mesoporous structure, a short incubation time of 5 min could be
300
adopted in enrichment process, which was superior to that of non-porous HILIC MNPs [7,
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9].
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The binding capacity of the hydrophilic MMNs toward HRP glycopeptides was
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investigated, and large binding capacity up to 200 mg/g was obtained (Fig. S3, see
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supporting information), which was comparable with the latest reported. [9] This might
305
be ascribed to the large surface area of hydrophilic MMNs.
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The post-enrichment recovery was investigated by using the proteolytic
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O labeling
method [32, 33]. As the mass spectra revealed (Fig. S4, see supporting information), the
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post-enrichment recovery of glycopeptides was up to 94.6% calculated according to the
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isotope distribution (based on relative intensity), which was higher than that of the latest
310
reported [8, 9].
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To investigate whether the hydrophilic MMNs can be recycled and reused for
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glycopeptide enrichment, the materials were regenerated by washing with the elution
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buffer several times. The regenerated MMNs were reused to enrich glycopeptides from
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HRP digest five times. As shown in Fig. S5 (see supporting information), the MS
315
spectrum of glycopeptides after enrichment five times was same as that for the first time,
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confirming the excellent run-to-run repeatability. In addition, the batch-to-batch
317
reproducibility of MMNs for glycopeptide enrichment was also assessed and the RSD
318
value was less than 5.2% (n=3), indicating that the MMNs can be synthesized reliably.
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3.3. Selective enrichment of glycopeptides from the digested mixture
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The applicability of the hydrophilic MMNs and Fe3O4@mSiO2–N3 MMNs (as a
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negative control) was further evaluated by isolation and enrichment of glycopeptides
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from a relatively complex digested mixture of HRP (10 pmol), myoglobin and β-casein
323
with a molar ratio of 1: 1: 10. Direct analysis revealed that nonglycopeptides dominated
324
the MS spectrum and only one glycopeptide at m/z 921.8924(2+) can be observed (Fig.
325
5A), which was due to the existence of abundant nonglycopeptides that suppressed the
326
ionization of low-abundance glycopeptides. After enrichment with the Fe3O4@mSiO2–
327
Glucose MMNs, although some hydrophilic nonglycopeptides were co-enriched in the
328
eluate, 20 peaks corresponding to 19 N-glycopeptides could still be observed (Fig. 5B).
329
Additionally, the peak intensities and S/N ratios of the identified N-glycopeptides
330
extracted by using Fe3O4@mSiO2–Glucose MMNs were also improved. In contrast, after
331
treatment with Fe3O4@mSiO2–N3 MMNs, only 2 peaks which belonged to the one
332
glycopeptide were detected for the same amount of sample (Fig. 5C), indicating that the
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immobilized click glucose greatly improved the hydrophilicity of MMNs.
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3.4. Selective enrichment of N-glycans from human serum
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To further challenge the enrichment capability of hydrophilic MMNs, glycans released
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from human serum were used as a complex sample. Human serum is of great importance
337
for biological functions in organs, and measurement of glycan changes of serum proteins
338
may provide relevant diagnostic and prognostic information [34]. Glycan enrichment is a
339
necessary step before MS analysis on account of the complexity and high dynamic range
340
of serum proteins. Recently, Zou’s group prepared the oxidized mesoporous carbon
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material [19] and mesoporous silica–carbon composite nanoparticles [20] for glycan
342
enrichment from serum samples. However, due to the distinct hydrophobicity of carbon,
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these carbon-based materials could simultaneously enrich endogenous peptides in serum,
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thus ultrafiltration procedure was required to remove serum peptides before the releasing
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and enrichment of N-linked glycans. [19,20] Herein, without the aid of ultrafiltration, the
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hydrophilic MMNs were employed to selectively extract N-linked glycans releasing from
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human serum by taking advantage of hydrophilicity of click glucose and size-exclusion to
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proteins.
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Fig. 6 presented the MS of N-glycans released from serum sample before and after
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hydrophilic MMNs enrichment. Before enrichment, only 12 N-glycans with low signal
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intensity could be identified (Fig. 6A). In contrast, obviously improved glycan
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identification was achieved after hydrophilic MMNs enrichment, where 42 N-glycans
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with largely increased signal intensity were identified from only 0.25 μL of the original
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human serum (Fig. 6B). The identified glycoforms were listed in Table S2 (see
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supporting information). The results of glycoform analysis indicated that all three N-
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glycoforms (high mannose, complex, and hybrid) were identified, demonstrating that
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nonbiased enrichment is achieved. In addition, the S/N of 4 of the identified glycans was
358
significantly enhanced more than 25-fold compared to direct analysis without enrichment
359
(see Table S3, supporting information), indicating the high efficiency for the enrichment
360
of glycans when using hydrophilic MMNs.
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4. Conclusions
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In summary, the novel core-shell structured MMNs, composed of superparamagnetic
363
core and click glucose modified mesoporous SiO2 shell, were synthesized for N-linked
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glycopeptide and glycan enrichment. The resulting Fe3O4@mSiO2–Glucose MMNs
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exhibited large external surface area and glycopeptide-suitable pore size, and excellent
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specificity towards glycopeptides and glycans. In addition, the excellent magnetic
367
response of the MMNs enabled the enrichment of glycopeptides and glycans to be very
368
convenient and rapid. Such hydrophilic MMNs are expected to be a promising affinity
369
material in glycoproteomic analysis.
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Acknowledgments
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This work was supported by the National Natural Science Foundation of China
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(21375018 and 21075016), National Basic Research Program of China (2010CB732403),
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the National Natural Science Funds for Distinguished Young Scholar (21125524), and
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the Program for Changjiang Scholars and Innovative Research Team in University (No.
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IRT1116).
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Captions
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Fig. 1 (A) Schematic representation of the synthesis of hydrophilic MMNs and (B) the
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selective enrichment process for glycopeptides using hydrophilic MMNs.
479
Fig. 2 SEM (A) and TEM (B) images of Fe3O4@mSiO2–Glucose MMNs; (C) Fe3O4
480
MNPs suspension in water; (D) Fe3O4@mSiO2–Glucose MMNs suspension in water (E)
481
Fe3O4@mSiO2–Glucose MMNs aggregation under magnetic field.
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Fig. 3 (A) N2 adsorption-desorption isotherm and pore size distribution curve of
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Fe3O4@mSiO2–Glucose MMNs; (B) FT-IR spectra of (a) Fe3O4@mSiO2–Cl with CTAB
484
templates, (b) Fe3O4@mSiO2–Cl, (c) Fe3O4@mSiO2–N3 and (b) Fe3O4@mSiO2–Glucose
485
MMNs; (C) XRD patterns of (a) Fe3O4 and (b) Fe3O4@mSiO2–Glucose MMNs; (D)
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Hysteresis loops of (a) Fe3O4 and (b) Fe3O4@mSiO2–Glucose MMNs.
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Fig. 4 ESI-Q-TOF mass spectra of 0.5 ng/μL tryptic HRP digestion. (A) Direct analysis
488
and (B) eluate from Fe3O4@mSiO2–Glucose MMNs after glycopeptide enrichment.
489
Peptide fragments containing N-glycan moiety are marked with stars.
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Fig. 5 ESI-Q-TOF mass spectra of the tryptic digest mixture of HRP, myoglobin and β-
491
casein with a molar ratio of 1: 1: 10. (A) Direct analysis; (B) eluate from Fe3O4@mSiO2–
492
Glucose MMNs after glycopeptide enrichment; (C) eluate from Fe3O4@mSiO2–N3
493
MMNs after glycopeptide enrichment. Peptide fragments containing N-glycan moiety are
494
marked with stars.
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Fig. 6 ESI-Q-TOF mass spectra of the N-Glycans identified from human serum PNGase
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F digest. (A) ultra-filter treated human serum PNGase F digest and (B) enrichment using
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Fe3O4@mSiO2–Glucose MMNs. Sialic acid ( ), galactose ( ), mannose ( ), GlcNAc
498
( ) and fucose ( ).
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Table 1. Detailed information of the glycopeptides detected from HRP digest Amino acid sequence [a]
Glycan composition
m/z with states
QSDQELFSSPN#ATDTIPLVR
Xyl1 Man3 GlcNAc2 Fuc1
848.3718(4+); 1130.4859(3+)
GLC(NQCR)PLNGN#LSALVDFDLR
Xyl1 Man3 GlcNAc2 Fuc1
NVGLN#R
Xyl1 Man3 GlcNAc2 Fuc1
921.9004(2+)
LHFHDCFVNGCDASILLDN#TTSFR
Xyl1 Man3 GlcNAc2 Fuc1
LHFHDCFVNGCDASILLDN#TTSFR
Xyl1 Man4 GlcNAc2 Fuc1
1015.1776(4+)
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Xyl1 Man3 GlcNAc2 Fuc1
1062.4463 (2+)
Xyl1 Man3 GlcNAc2
1087.4339 (2+)
M
SFAN#STQTFF
902.6596(4+); 1203.2057(3+)
us
LDN#TTSFR
LDN#TTSFR
charge
974.6653(4+); 1299.5421(3+)
an
499
Xyl1 Man4 GlcNAc2 Fuc1
1143.9877 (2+)
Xyl1 Man3 GlcNAc2 Fuc1
1160.9726 (2+)
GLIQSDQELFSSPN#ATDTIPLVR
Xyl1 Man3 GlcNAc2 Fuc1
1224.5780(3+)
LHFHDCFVNGCDASILLDN#TTSF
Xyl1 Man3 GlcNAc2 Fuc1
1247.5262(3+)
SSPN#ATDTIPLVR
Xyl1 Man3 GlcNAc2 Fuc1
1271.5731(2+)
QLTPTFYDN#SC(AAVESACPR)PNVSNIVR- Xyl1 Man3 GlcNAc2 Fuc1 H2O
1408.6138(3+)
LYN#FSNTGLPDPTLN#TTY
1458.6135 (3+)
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SFAN#STQTFF
Xyl1 Man3 GlcNAc2 Fuc1 Xyl1 Man3 GlcNAc2 Fuc1
LYN#FSNTGLPDPTLN#TTYL
Xyl1 Man3 GlcNAc2 Fuc1
1496.3058 (3+)
Xyl1 Man3 GlcNAc2 Fuc1
LYN#FSNTGLPDPTLN#TTYLQTLR
Xyl1 Man3 GlcNAc2 Fuc1
1662.4072(3+)
Xyl1 Man3 GlcNAc2 Fuc1 500
[a] The N–glycosylation sites are marked with N#. GlcNAc = N–acetylglucosamine, Fuc
501
= fructose, Man = mannose, Xyl= xylose.
502 24
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Highlights
503 504
(1) Hydrophilic magnetic mesoporous nanoparticles were synthesized via click chemistry.
505 506
(2) The mesoporous nanoparticles showed high hydrophilicity and large surface area.
507 508
(3) Successful application to the selective enrichment of glycopeptides from protein digests.
509 510
(4) Successful application to the selective enrichment of glycans from digested human serum.
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511 512
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S0021-9673(14)01013-9 http://dx.doi.org/doi:10.1016/j.chroma.2014.06.070 CHROMA 355549
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
18-12-2013 11-6-2014 22-6-2014
Please cite this article as: J. Zheng, Y. Xiao, L. Wang, Z. [email protected], H. Yang, L. [email protected], G. Chen, Click synthesis of glucosefunctionalized hydrophilic magnetic mesoporous nanoparticles for highly selective enrichment of glycopeptides and glycans, Journal of Chromatography A (2014), http://dx.doi.org/10.1016/j.chroma.2014.06.070 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Click synthesis of glucose-functionalized hydrophilic magnetic
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mesoporous nanoparticles for highly selective enrichment of
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glycopeptides and glycans
4 5
Jiangnan Zheng, Yun Xiao, Ling Wang, Zian Lin,* Huanghao Yang, Lan Zhang,*
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and Guonan Chen
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Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian
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Provincial Key Laboratory of Analysis and Detection Technology for Food Safety,
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Department of Chemistry, Fuzhou University, Fuzhou, Fujian, 350108, China
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First corresponding author: Zian Lin;
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Second corresponding author: Lan Zhang
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Postal address: Department of Chemistry, Fuzhou University,
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Fuzhou, Fujian, 350108, China
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Fax: 86-591-22866135
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E-mail: [email protected] (Z.A. Lin); [email protected] (L.Zhang);
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Abstract 1
Page 1 of 31
Selective enrichment of glycopeptides from complex biological samples is essential for
24
MS-based glycoproteomics, but challenges still remain. In this work, glucose-
25
functionalized magnetic mesoporous nanoparticles (MMNs), which hold the attractive
26
features of well-defined core/shell structure, high specific surface area (324 m2/g), narrow
27
pore size distribution (2.2 nm) and high magnetic responsivity (69.1 emu/g), were
28
synthesized via click chemistry and applied to enrich glycopeptides and glycans. Taking
29
advantages of the size-exclusive effect of mesopore against proteins and the hydrophilic
30
interaction between glycans and glucose, the hydrophilic MMNs possessed high
31
selectivity for glycopeptides at the digested mixtures of horseradish peroxidase (HRP),
32
myoglobin and β-casein at molar ratio of 1:1:10, large enrichment capacity (as high as
33
250 mg/g), high sensitivity (50 fmol), excellent speed (5 min for enrichment) and high
34
recovery of glycopeptides (as high as 94.6%). Additionally, the MMNs exhibited
35
excellent performance in enrichment of N-linked glycans from the digested human serum
36
that are made up of peptides, large proteins and other compounds. These outstanding
37
features will give the hydrophilic MMNs high benefit for MS analysis of low-abundance
38
glycopeptides/glycans.
39
Keywords:
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glycopeptide, glycan, enrichment
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magnetic
mesoporous
nanoparticles,
click
chemistry,
hydrophilic,
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1. Introduction
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Protein glycosylation, as one of the most ubiquitous and complex post-translational
46
modifications, plays a vital role in many biological processes [1,2]. Numerous studies
47
have revealed that many clinical biomarkers are glycoproteins. Currently, mass
48
spectrometry (MS)-based technique has been a preferred choice in glycoproteome
49
analysis. However, owing to the inherent low abundance and poor ionization efficiency
50
of glycopeptides, enrichment and identification of glycopeptides is still an enduring
51
challenge. In the past few years, considerable efforts have been devoted to developing
52
methods for isolation and enrichment of glycopeptides including lectin-based affinity
53
chromatography [3], boronate affinity chromatography [4], hydrazine chemistry [5],
54
titanium dioxide [6], and hydrophilic interaction chromatography (HILIC) [7-9]. Among
55
these methods, HILIC have attracted wide interest because of its good reproducibility,
56
broad glycan-binding specificity, and excellent compatibility with MS analysis.
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Several hydrophilic absorbents/stationary phases have been widely used in recent years
58
for glycoprotein/glycopeptide enrichment, including sepharose [10], cellulose [11],
59
zwitterionic monomers [12], and maltose [13, 14]. Recently, Xiong et al. prepared
60
branched PEG brushes hybrid magnetic nanoparticles (MNPs) and then the reactive
61
hydroxyl groups were modified with maltose, which could increase the amount of
62
immobilized maltose and provide high selectivity and sensitivity for glycopeptide
63
detection[8]. However, the synthesis processes were tedious. More recently, Xiong et al.
64
fabricated multilayer polysaccharide shells coated MNPs using a layer-by-layer (LBL)
65
approach via the alternate deposition of hyaluronan (HA) and chitosan (CS) onto the
66
surface, and the MNPs-(HA/CS)10 exhibited high selectivity, high detection sensitivity,
67
large binding capacity for the enrichment of glycopeptides[9]. Yet, the enrichment
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recovery of MNPs (69%) needs to be improved. And the LbL-assembled nanostructure
69
may suffer from a lack of stability when they are exposed to certain changes in aqueous
70
environments [15]. Therefore, developing an effective and robust HILIC method for
71
glycopeptide enrichment is highly desirable.
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Ordered mesoporous materials have attracted immense interest in the fields of
73
nanosensor, catalyst, and biomedical engineering due to the large surface area, well-
74
defined mesoporous structure, and narrow pore size distribution [16]. The application of
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mesoporous materials in biological sample pretreatment, such as selective capture of
76
endogenous peptides [17], phosphopeptides [18] and glycans [19,20], has been reported
77
recently, where the unique mesoporous structures were superior in capturing target
78
peptides and repelling against interfering large molecular weight proteins due to the size-
79
exclusion effect. Meanwhile, magnetic materials have received great attention due to their
80
magnetism properties, and have been applied extensively in proteomics, including protein
81
digestion and proteins/peptides enrichment [21-25]. More recently, taking advantages of
82
magnetic materials and mesoporous structure, Lu et al. [26] developed a novel method to
83
synthesize magnetic mesoporous Fe3O4@mTiO2 microspheres with high surface area,
84
large pore volume, and high magnetic susceptibility, which possessed remarkable
85
selectivity and high enrichment capacity for phosphopeptides. Nevertheless, no reports on
86
hydrophilic magnetic mesoporous materials for selective isolation and enrichment of
87
glycopeptides and glycans have been found so far.
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Herein, we report a facile approach for the synthesis of glucose-functionalized
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hydrophilic magnetic mesoporous nanoparticles (MMNs) via click reaction and applied
90
them for selective isolation and enrichment of glycopeptides/glycans. The hydrophilic
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MMNs hold the features of high surface area and glycopeptide-suitable pore size, and
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thus can be employed to enrich glycopeptides/glycans from complex biological samples.
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Besides, with the unique magnetic responsivity, they can be easily removed from the
94
mixture by employing an external magnet, rather than with the help of centrifugation.
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This proposed approach offered higher efficiency in enrichment of glycopeptides/glycans
96
than the previous reported methods.
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2. Experimental
98
2.1. Chemicals and reagents
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Sodium citrate dehydrate (Na3Cit·2H2O), ethylene glycol (EG), and copper (II) sulphate
100
pentahydrate (CuSO4·5H2O) were obtained from Sinopharm Chemical Reagent, Co., Ltd
101
(Shanghai, China). Iron (III) chloride (FeCl3), sodium ascorbate, tetraethylorthosilicate
102
(TEOS), 3-chloropropyltriethoxysilane (CPTES), D-glucose, zinc dust (99.99%),
103
cetyltrimethylammonium bromide (CTAB), formic acid (FA) and sequencing-grade
104
modified trypsin (TPCK-trypsin) were obtained from Aladdin Chemistry Co., Ltd
105
(Shanghai, China). Propargyl bromide (80 wt% solution in toluene) was purchased from
106
J&K Chemical Ltd (Shanghai, China). HRP was purchased from Shanghai Lanji Co. Ltd.
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(Shanghai, China). Sodium azide (NaN3), horse myoglobin (Mb, Sigma cat. no. M1882),
108
and β-casein from bovine milk (Sigma C6905) were the products of Sigma-Aldrich (St.
109
Louis, USA). Peptide N-glycosidase (PNGase F) was purchased from New England
110
Biolabs (Ipswi ch, MA, USA). Centrifugal filter with MWCO of 10 kDa was purchased
111
from Millipore (Bedford, MA). Deionized water was prepared with a Milli-Q water
112
purification system (Millipore, Milford, MA). All other chemicals and reagents were of
113
analytical grade or better.
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2.2. Preparation of Fe3O4 magnetic nanoparticles The magnetic nanoparticles (MNPs) were prepared according to the previous
116
description [27] with some modifications. Briefly, FeCl3 (6.8 g), sodium acetate (12.0 g),
117
Na3Cit·2H2O (2.0 g) were dissolved in EG (200 mL). The obtained homogeneous yellow
118
solution was transferred to autoclave, and then heated to 200 °C for 7 h. After reaction,
119
the product was collected with a magnet and washed with water and ethanol for several
120
times and then dried at room temperature.
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2.3. Preparation of Fe3O4@mSiO2-Cl MMNs
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The synthesized Fe3O4 MNPs were coated with mesoporous silica shells by hydrolysis
123
and condensation of TEOS and CPTES. Typically, the Fe3O4 nanoparticles (0.5 g) and
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CTAB (1.0 g) were added into 500 mL H2O, and ultrasonically dispersed for 1 h to form
125
a stable dispersion. Afterward, 80 mL of NaOH aqueous solution (7.5 mM) was added to
126
above mixture and further stirred at 60 °C for another 30 min. Then, 20 mL of
127
TEOS/CPTES/ethanol (v/v/v: 2/1/2) solution was added by injection under mechanical
128
stirring, and subsequently the dispersion was further heated at 60 °C for 12 h. The
129
obtained product (Fe3O4@SiO2) was collected by magnetic separation and washed with
130
ethanol repeatedly, and redispersed in ethanol (60 mL) for refluxing at 90 °C to remove
131
the CTAB. The refluxing process was repeated 5 times to achieve a complete removal of
132
CTAB, and the resulting Fe3O4@mSiO2–Cl MMNs were dried at 50 °C for 24 h for the
133
future use.
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2.4. Preparation of Fe3O4@mSiO2-N3 MMNs
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Fe3O4@mSiO2-Cl MMNs (0.5 g) were dispersed in 100 mL saturated solution of NaN3
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in DMF. The obtained mixture was stirred at 80°C for 24 h. After reaction, the obtained
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137
product (Fe3O4@mSiO2-N3) was collected by magnetic separation, washed with ethanol
138
repeatedly, and dried at room temperature.
139
2.5. Synthesis of propargyl glucose Propargyl glucose was obtained through direct addition of propargyl bromide to
141
aldehyde substrates mediated with low valent iron [28]. Briefly, a mixture of D-glucose
142
(0.90 g, 5 mmol), propargyl bromide (1.6 mL, 80 wt% solution in toluene, 15 mmol), and
143
FeCl3 (2.43 g, 15 mmol) in THF (100 mL) was stirred thoroughly in a water bath at
144
around 10~15 °C (maintained by addition of pieces of ice). After stirring the mixture for
145
10 min, zinc dust (0.975 g, 15 mmol) was added portion wise over a period of 8 min. The
146
mixture was stirred at room temperature for 12h. After reaction, the mixture was then
147
treated successively with diethyl ether (100 mL) and water (50 mL), stirred for 10 min
148
and then filtered. The filtrate was treated with ammonium bicarbonate to precipitate metal
149
ions and then filtered. The aqueous layer was used without drying.
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2.6. Preparation of Fe3O4@mSiO2-Glucose MMNs
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Fe3O4@mSiO2-N3 MMNs (0.5 g) was dispersed in 100 mL H2O. Then, the propargyl
152
glucose solution (in 100 mL water) was added to the above mixture, followed by
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CuSO4·5H2O (372 mg, 1.5 mmol) and sodium ascorbate (892 mg, 4.5 mmol). The
154
reaction mixture stirred in air at RT for 12 h. After reaction, the product was washed with
155
water and ethanol for several times and dried at room temperature.
156
2.7. Characterization
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Scanning electron microscopy (SEM) images were obtained with FEI Inspect F50 (FEI,
158
USA). Transmission electron microscopy (TEM) analysis was performed on a FEI Tecnai
159
G2 20 (FEI, USA) at 200 kV. Nitrogen adsorption and desorption isotherms were
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measured using an ASAP 2020 (Micromeritics, USA). The samples were degassed in a
161
vacuum at 200 °C for 8 h prior to measurement. Brunauer–Emmett–Teller (BET) method
162
was utilized to calculate the specific surface areas (SBET) using adsorption data in a
163
relative pressure range from 0.18 to 0.35. By using the Barrett-Joyner-Halenda (BJH)
164
model, the pore volume and pore size distribution were derived from the adsorption
165
branches of the isotherms, and the total pore volumes were estimated from the adsorbed
166
amount at a relative pressure P/P0 of 0.992. Fourier-transform infrared (FT-IR) spectra
167
were taken on Nicolet 6700 spectrometer (Thermo Fisher, USA) using KBr pellets. The
168
crystal structure of the MMNs was determined by X'Pert-Pro MPD (Philips, Holland).
169
The magnetization curves were carried out at room temperature on the superconducting
170
quantum interference device magnetometer (SQUID) MPMS XL-7 (Quantum Design,
171
San Diego, USA).
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2.8. Release of N-Linked glycans from human serum
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To release the N-linked glycans, human serum were thawed at 4 ºC and centrifuged at
174
13000g for 10 min. The collected supernatants (50 μL) were diluted to 500 μL by 10 mM
175
ammonium bicarbonate (pH 7.5). Then the solutions (100 μL) were boiled for 5 min,
176
cooled to room temperature, and incubated at 37 ºC for 24 h after adding 10 U of PNGase
177
F.
178
2.9. Enrichment of N-Linked glycopeptides and glycans
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Tryptic digests of protein were dissolved in 200 μL binding buffer (80% ACN
180
containing 0.2% FA). Then, 60 μg Fe3O4@mSiO2–Glucose MMNs were suspended in
181
above solution in a microcentrifuge tube. After incubated for 5 min, the MMNs having
182
captured the glycopeptides were isolated from the solution with an assistant magnet and
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183
washed three times with binding buffer in order to remove residual uncaptured peptides.
184
Finally, the captured peptides were eluted with 10 μL elution buffer (20% ACN
185
containing 0.2% FA) for 5 min, and the eluate was analyzed by ESI-Q-TOF MS. To enrich the N-linked glycans, the diluted serum digestion (2.5 μL) was dissolved in
187
400 μL binding buffer (80% ACN containing 0.2% FA). Then, the Fe3O4@mSiO2–
188
Glucose MMNs (200 μg) were suspended in the solution to enrich glycans. The other
189
procedures for the enrichment of N-linked glycans from human serum were the same as
190
for the above mentioned glycopeptides.
191
2.10. Mass spectrometry
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A 1200 series HPLC system with a binary SL pump was used. Detection was
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performed using an Agilent 6520 Q-TOF with dual electrospray ion source (ESI). The
194
separation of all samples was performed on Agilent Poroshell 120 EC-C18 column
195
(2.7 μm, 3.0 mm × 50 mm, Agilent). The flow rate was 0.3 mL/min. Solvent A was
196
composed of water containing 0.1% formic acid. Solvent B was composed of ACN
197
containing 0.1% formic acid. The following gradient program was used: 0–3 min, 3% B;
198
3–20 min, 3–40% B; 20–23 min, 40–80% B; 23–25 min, 80% B. The sample injection
199
volume was 3 μL. Nitrogen was used as drying gas at a temperature of 350°C and a flow–
200
rate of 10 L/min. Full scan MS data and MS/MS data were acquired at m/z 800-2000 and
201
100-3000, respectively. Scan rates for MS and MS/MS data were set to 3 spectra/sec. The
202
voltage set for the MS capillary was 4 kV and the fragmentor was set to 175 V. For MS2
203
experiments, the collision energy was set to according to formula (see supporting
204
information). GlycoWorkbench [29] software was applied for mass spectrometric data
205
interpretation and glycoform analysis.
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3. Results and discussion
207
3.1. Synthesis and characterization of hydrophilic MMNs The general scheme for the synthesis of hydrophilic MMNs is illustrated in Fig. 1.
209
Firstly, using CTAB as a structural directing agent, TEOS and CPTES as the precursors,
210
a CTAB/mSiO2–Cl composite was deposited on the Fe3O4 MNPs by sol-gel process.
211
Secondly, CTAB templates were removed by ethanol extraction to form a mesoporous
212
SiO2 shell, resulting in well-dispersed Fe3O4@mSiO2–Cl MMNs. Thirdly, the
213
chloropropylated MMNs were treated with NaN3 to obtain Fe3O4@mSiO2–N3 MMNs.
214
Finally, Fe3O4@mSiO2–Glucose MMNs were synthesized by click reaction between
215
Fe3O4@mSiO2–N3 MMNs and propargyl glucose.
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Representative SEM and TEM images showed that the synthesized Fe3O4@mSiO2–
217
Glucose MMNs were approximately spherical in shape with a core-shell structure, which
218
consisted of a magnetic core (ca. 200 nm) and a thin mesoporous SiO2 shell (ca. 15 nm)
219
with perpendicularly oriented channels (Fig. 2A and B). The short channels of
220
Fe3O4@mSiO2–Glucose MMNs might improve the mass transfer efficiency during
221
enrichment of low molecular weight (MW) biomolecules [20]. The magnetic core with
222
narrow size distribution was obtained by means of a solvothermal reaction, and could
223
well disperse in aqueous solution (Fig. 2C). After sol-gel process, the color of the MMNs
224
changed from black to bright orange (Fig. 2D), indicating the successful coating of silica
225
shell as well. The functionalized MMNs also exhibited high dispersibility in aqueous
226
phase.
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The structural properties of the Fe3O4@mSiO2–Glucose MMNs were studied by
228
nitrogen adsorption-desorption at 77 K, as shown in Fig. 3A. The Fe3O4@mSiO2–
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Glucose MMNs exhibited a type IV isotherm, which was typical for mesoporous silica
230
synthesized with CTAB as surfactant. From pore size distribution curve, it was observed
231
that the pore diameter of the MMNs was about 2.2 nm and displayed a narrow size
232
distribution. The BET surface area and total pore volume were calculated to be 324 m2/g
233
and 0.25 cm3/g, respectively. Taking together, these results showed that the core-shell
234
structure of Fe3O4@mSiO2–Glucose MMNs had a large specific surface area and large
235
pore volume, which was caused by the existence of mesoporous silica coating.
236
Significantly, the high degree of porosity and suitable mesopore size of these MMNs
237
made it possible to selectively extract low MW biomolecules from complex samples.
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FT-IR spectra (Fig.3B) provided a direct proof for synthetic process of the
239
Fe3O4@mSiO2–Glucose MMNs. The spectrum of Fe3O4@mSiO2–Cl MMNs with CTAB
240
templates was showed in curve a. The typical absorption bands at 584 and 1080 cm−1
241
could be assigned to Fe–O–Fe and Si–O–Si vibrations, respectively. Moreover, the strong
242
absorption peak at 2925 and 2850 cm−1 assigned to –CH2 groups indicated the successful
243
coating of CTAB-templated silica layer. After refluxing process, the absorption peak of –
244
CH2 became weak (curve b), confirming the removal of CATB. Compared to spectrum b,
245
the appearance of a characteristic peak at 2104 cm−1 due to the out-of-phase stretching
246
vibration of azido groups in spectrum of Fe3O4@mSiO2–N3 MMNs (curve c) clearly
247
suggested that the chlorine atoms had been substituted by N3 groups. The spectrum of
248
Fe3O4@mSiO2–Glucose MMNs (curve d) showed the disappearance of the N3 vibration
249
band at 2104 cm−1, which indicated all N3 groups did react with propargyl glucose by
250
virtue of a highly efficient alkyne-azide click reaction [30]. Meanwhile, the band
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251
intensity of –CH2 groups at 2945 and 2875 cm−1 was increased after click reaction, which
252
also implied the successful surface modification of MMNs with glucose. The crystalline structures of Fe3O4 and Fe3O4@mSiO2–Glucose MMNs were
254
determined by XRD (Fig. 3C). The positions and relative intensities of all diffraction
255
peaks for Fe3O4 and Fe3O4@mSiO2–Glucose MMNs matched well with those from the
256
JCPDS card (19-629) for magnetite. The broad diffraction peaks suggested the magnetite
257
core was composed of small nanocrystals [27]. The size of these small nanocrystals,
258
calculated from the Debye-Scherrer formula for the strongest (311) diffraction peak, was
259
11.8 nm. The unique structure allowed MMNs to retain superparamagnetic behaviour at
260
room temperature even though their size exceeded 30 nm. The magnetic characterization
261
on a SQUID (Fig. 3D) confirmed that both of the bare Fe3O4 MNPs and Fe3O4@mSiO2–
262
Glucose MMNs exhibited superparamagnetic behavior at 300K. No remanence remained
263
when the applied magnetic field was removed. Their corresponding saturated
264
magnetization values were 79.8 and 69.1 emu g−1, respectively. Such high saturation
265
magnetization endued the MMNs with a fast response to an external magnetic field. (Fig.
266
2D and E). It was suggested that the MMNs possessed excellent magnetic responsiveness.
267
3.2. Selective enrichment of glycopeptides from HRP digest
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268
To evaluate the selectivity and enrichment efficiency of the Fe3O4@mSiO2–Glucose
269
MMNs, tryptic HRP digest was employed as a model sample. It is well known that HRP
270
has nine potential glycosylation sites of which at least eight sites are occupied by
271
heterogeneous high-mannose type oligosaccharides [31]. It is generally acceptable that
272
glycopeptides would be bound more strongly than nonglycopeptides on Fe3O4@mSiO2–
273
Glucose MMNs based on the hydrophilic interactions between glucose and glycan
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274
moieties. In order to make the hydrophilic interaction play a dominant role for
275
glycopeptide enrichment, ACN/H2O/FA (80/19.8/0.2, v/v/v) was recommended as
276
loading buffer. Fig. 4 showed the direct MS analysis of 0.5 ng/μL HRP tryptic digest treated with and
278
without Fe3O4@mSiO2–Glucose MMNs. Without treatment, only 4 peaks that matched
279
the glycopeptides (the identified N-glycopeptides was displayed in Table S1, see
280
supporting information) could be determined from 0.5 ng/μL tryptic HRP digestion (Fig.
281
4A). In contrast, after treatment with the MMNs, up to 20 peaks corresponding to 18 N-
282
glycopeptides could be clearly detected with higher signal to noise ratios (S/N) (Fig. 4B).
283
The oligosaccharide composition and amino sequence of these detected glycopeptides
284
(Table 1) was in agreement with the previous report describing a xylosylated, core-(α1-
285
3)-fucosylated trimannosyl N-glycan structure as the predominant species at all N-
286
glycosylation sites of HRP [31]. Furthermore, seven HRP peptides of unreported
287
structure were confirmed as glycopeptides according to their product ions at m/z
288
163.0601 (Man+), m/z 204.0866 (GlcNAc+), m/z 366.1394 (Man1GlcNAc1+), m/z
289
528.1923
290
(Man3GlcNAc1+) and 822.2873 (XylMan3GlcNAc1+), which were marker oxonium ions
291
of glycopeptides (Fig. S1, see supporting information). Furthermore, the sensitivity was
292
further evaluated by the enrichment of glycopeptides from low concentration of HRP
293
digest (0.011 ng/μL, corresponding to 50 fmol HRP), two glycopeptides were still
294
observed after enrichment (Fig. S2, see supporting information). The results indicated
295
that the Fe3O4@mSiO2–Glucose MMNs had predominant enrichment efficiency and high
296
selectivity for the glycopeptides. Totally 8 N-glycosylation sites (Table 1) of HRP were
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(Man2GlcNAc1+),
m/z
660.2345
13
(XylMan2GlcNAc1+),
m/z
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Page 13 of 31
identified by the hydrophilic MMNs, which exhibited better enrichment performance than
298
PEG-Maltose hybrid MNPs (6 identified glycosylation sites of HRP) [8]. Moreover,
299
owing to the unique mesoporous structure, a short incubation time of 5 min could be
300
adopted in enrichment process, which was superior to that of non-porous HILIC MNPs [7,
301
9].
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The binding capacity of the hydrophilic MMNs toward HRP glycopeptides was
303
investigated, and large binding capacity up to 200 mg/g was obtained (Fig. S3, see
304
supporting information), which was comparable with the latest reported. [9] This might
305
be ascribed to the large surface area of hydrophilic MMNs.
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The post-enrichment recovery was investigated by using the proteolytic
18
O labeling
method [32, 33]. As the mass spectra revealed (Fig. S4, see supporting information), the
308
post-enrichment recovery of glycopeptides was up to 94.6% calculated according to the
309
isotope distribution (based on relative intensity), which was higher than that of the latest
310
reported [8, 9].
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To investigate whether the hydrophilic MMNs can be recycled and reused for
312
glycopeptide enrichment, the materials were regenerated by washing with the elution
313
buffer several times. The regenerated MMNs were reused to enrich glycopeptides from
314
HRP digest five times. As shown in Fig. S5 (see supporting information), the MS
315
spectrum of glycopeptides after enrichment five times was same as that for the first time,
316
confirming the excellent run-to-run repeatability. In addition, the batch-to-batch
317
reproducibility of MMNs for glycopeptide enrichment was also assessed and the RSD
318
value was less than 5.2% (n=3), indicating that the MMNs can be synthesized reliably.
319
3.3. Selective enrichment of glycopeptides from the digested mixture
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The applicability of the hydrophilic MMNs and Fe3O4@mSiO2–N3 MMNs (as a
321
negative control) was further evaluated by isolation and enrichment of glycopeptides
322
from a relatively complex digested mixture of HRP (10 pmol), myoglobin and β-casein
323
with a molar ratio of 1: 1: 10. Direct analysis revealed that nonglycopeptides dominated
324
the MS spectrum and only one glycopeptide at m/z 921.8924(2+) can be observed (Fig.
325
5A), which was due to the existence of abundant nonglycopeptides that suppressed the
326
ionization of low-abundance glycopeptides. After enrichment with the Fe3O4@mSiO2–
327
Glucose MMNs, although some hydrophilic nonglycopeptides were co-enriched in the
328
eluate, 20 peaks corresponding to 19 N-glycopeptides could still be observed (Fig. 5B).
329
Additionally, the peak intensities and S/N ratios of the identified N-glycopeptides
330
extracted by using Fe3O4@mSiO2–Glucose MMNs were also improved. In contrast, after
331
treatment with Fe3O4@mSiO2–N3 MMNs, only 2 peaks which belonged to the one
332
glycopeptide were detected for the same amount of sample (Fig. 5C), indicating that the
333
immobilized click glucose greatly improved the hydrophilicity of MMNs.
334
3.4. Selective enrichment of N-glycans from human serum
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335
To further challenge the enrichment capability of hydrophilic MMNs, glycans released
336
from human serum were used as a complex sample. Human serum is of great importance
337
for biological functions in organs, and measurement of glycan changes of serum proteins
338
may provide relevant diagnostic and prognostic information [34]. Glycan enrichment is a
339
necessary step before MS analysis on account of the complexity and high dynamic range
340
of serum proteins. Recently, Zou’s group prepared the oxidized mesoporous carbon
341
material [19] and mesoporous silica–carbon composite nanoparticles [20] for glycan
342
enrichment from serum samples. However, due to the distinct hydrophobicity of carbon,
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these carbon-based materials could simultaneously enrich endogenous peptides in serum,
344
thus ultrafiltration procedure was required to remove serum peptides before the releasing
345
and enrichment of N-linked glycans. [19,20] Herein, without the aid of ultrafiltration, the
346
hydrophilic MMNs were employed to selectively extract N-linked glycans releasing from
347
human serum by taking advantage of hydrophilicity of click glucose and size-exclusion to
348
proteins.
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Fig. 6 presented the MS of N-glycans released from serum sample before and after
350
hydrophilic MMNs enrichment. Before enrichment, only 12 N-glycans with low signal
351
intensity could be identified (Fig. 6A). In contrast, obviously improved glycan
352
identification was achieved after hydrophilic MMNs enrichment, where 42 N-glycans
353
with largely increased signal intensity were identified from only 0.25 μL of the original
354
human serum (Fig. 6B). The identified glycoforms were listed in Table S2 (see
355
supporting information). The results of glycoform analysis indicated that all three N-
356
glycoforms (high mannose, complex, and hybrid) were identified, demonstrating that
357
nonbiased enrichment is achieved. In addition, the S/N of 4 of the identified glycans was
358
significantly enhanced more than 25-fold compared to direct analysis without enrichment
359
(see Table S3, supporting information), indicating the high efficiency for the enrichment
360
of glycans when using hydrophilic MMNs.
361
4. Conclusions
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In summary, the novel core-shell structured MMNs, composed of superparamagnetic
363
core and click glucose modified mesoporous SiO2 shell, were synthesized for N-linked
364
glycopeptide and glycan enrichment. The resulting Fe3O4@mSiO2–Glucose MMNs
365
exhibited large external surface area and glycopeptide-suitable pore size, and excellent
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specificity towards glycopeptides and glycans. In addition, the excellent magnetic
367
response of the MMNs enabled the enrichment of glycopeptides and glycans to be very
368
convenient and rapid. Such hydrophilic MMNs are expected to be a promising affinity
369
material in glycoproteomic analysis.
370
Acknowledgments
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This work was supported by the National Natural Science Foundation of China
372
(21375018 and 21075016), National Basic Research Program of China (2010CB732403),
373
the National Natural Science Funds for Distinguished Young Scholar (21125524), and
374
the Program for Changjiang Scholars and Innovative Research Team in University (No.
375
IRT1116).
376
References
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[4] Y.Y. Qu, J.X. Liu, K.G. Yang, Z. Liang, L.H. Zhang, Y.K. Zhang, Boronic acid
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functionalized core–shell polymer nanoparticles prepared by distillation precipitation
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Captions
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Fig. 1 (A) Schematic representation of the synthesis of hydrophilic MMNs and (B) the
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selective enrichment process for glycopeptides using hydrophilic MMNs.
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Fig. 2 SEM (A) and TEM (B) images of Fe3O4@mSiO2–Glucose MMNs; (C) Fe3O4
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MNPs suspension in water; (D) Fe3O4@mSiO2–Glucose MMNs suspension in water (E)
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Fe3O4@mSiO2–Glucose MMNs aggregation under magnetic field.
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Fig. 3 (A) N2 adsorption-desorption isotherm and pore size distribution curve of
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Fe3O4@mSiO2–Glucose MMNs; (B) FT-IR spectra of (a) Fe3O4@mSiO2–Cl with CTAB
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templates, (b) Fe3O4@mSiO2–Cl, (c) Fe3O4@mSiO2–N3 and (b) Fe3O4@mSiO2–Glucose
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MMNs; (C) XRD patterns of (a) Fe3O4 and (b) Fe3O4@mSiO2–Glucose MMNs; (D)
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Hysteresis loops of (a) Fe3O4 and (b) Fe3O4@mSiO2–Glucose MMNs.
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Fig. 4 ESI-Q-TOF mass spectra of 0.5 ng/μL tryptic HRP digestion. (A) Direct analysis
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and (B) eluate from Fe3O4@mSiO2–Glucose MMNs after glycopeptide enrichment.
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Peptide fragments containing N-glycan moiety are marked with stars.
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Fig. 5 ESI-Q-TOF mass spectra of the tryptic digest mixture of HRP, myoglobin and β-
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casein with a molar ratio of 1: 1: 10. (A) Direct analysis; (B) eluate from Fe3O4@mSiO2–
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Glucose MMNs after glycopeptide enrichment; (C) eluate from Fe3O4@mSiO2–N3
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MMNs after glycopeptide enrichment. Peptide fragments containing N-glycan moiety are
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marked with stars.
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Fig. 6 ESI-Q-TOF mass spectra of the N-Glycans identified from human serum PNGase
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F digest. (A) ultra-filter treated human serum PNGase F digest and (B) enrichment using
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Fe3O4@mSiO2–Glucose MMNs. Sialic acid ( ), galactose ( ), mannose ( ), GlcNAc
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( ) and fucose ( ).
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Table 1. Detailed information of the glycopeptides detected from HRP digest Amino acid sequence [a]
Glycan composition
m/z with states
QSDQELFSSPN#ATDTIPLVR
Xyl1 Man3 GlcNAc2 Fuc1
848.3718(4+); 1130.4859(3+)
GLC(NQCR)PLNGN#LSALVDFDLR
Xyl1 Man3 GlcNAc2 Fuc1
NVGLN#R
Xyl1 Man3 GlcNAc2 Fuc1
921.9004(2+)
LHFHDCFVNGCDASILLDN#TTSFR
Xyl1 Man3 GlcNAc2 Fuc1
LHFHDCFVNGCDASILLDN#TTSFR
Xyl1 Man4 GlcNAc2 Fuc1
1015.1776(4+)
ip t
cr
Xyl1 Man3 GlcNAc2 Fuc1
1062.4463 (2+)
Xyl1 Man3 GlcNAc2
1087.4339 (2+)
M
SFAN#STQTFF
902.6596(4+); 1203.2057(3+)
us
LDN#TTSFR
LDN#TTSFR
charge
974.6653(4+); 1299.5421(3+)
an
499
Xyl1 Man4 GlcNAc2 Fuc1
1143.9877 (2+)
Xyl1 Man3 GlcNAc2 Fuc1
1160.9726 (2+)
GLIQSDQELFSSPN#ATDTIPLVR
Xyl1 Man3 GlcNAc2 Fuc1
1224.5780(3+)
LHFHDCFVNGCDASILLDN#TTSF
Xyl1 Man3 GlcNAc2 Fuc1
1247.5262(3+)
SSPN#ATDTIPLVR
Xyl1 Man3 GlcNAc2 Fuc1
1271.5731(2+)
QLTPTFYDN#SC(AAVESACPR)PNVSNIVR- Xyl1 Man3 GlcNAc2 Fuc1 H2O
1408.6138(3+)
LYN#FSNTGLPDPTLN#TTY
1458.6135 (3+)
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SFAN#STQTFF
Xyl1 Man3 GlcNAc2 Fuc1 Xyl1 Man3 GlcNAc2 Fuc1
LYN#FSNTGLPDPTLN#TTYL
Xyl1 Man3 GlcNAc2 Fuc1
1496.3058 (3+)
Xyl1 Man3 GlcNAc2 Fuc1
LYN#FSNTGLPDPTLN#TTYLQTLR
Xyl1 Man3 GlcNAc2 Fuc1
1662.4072(3+)
Xyl1 Man3 GlcNAc2 Fuc1 500
[a] The N–glycosylation sites are marked with N#. GlcNAc = N–acetylglucosamine, Fuc
501
= fructose, Man = mannose, Xyl= xylose.
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Highlights
503 504
(1) Hydrophilic magnetic mesoporous nanoparticles were synthesized via click chemistry.
505 506
(2) The mesoporous nanoparticles showed high hydrophilicity and large surface area.
507 508
(3) Successful application to the selective enrichment of glycopeptides from protein digests.
509 510
(4) Successful application to the selective enrichment of glycans from digested human serum.
us
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