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One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
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One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides Chunli Fang a , Zhichao Xiong b , Hongqiang Qin c , Guang Huang c, Jing Liu c , Mingliang Ye c, Shun Feng a, * , Hanfa Zou c, ** a Key Laboratory of Oil Gas Fine Chemicals, Ministry of Education Xinjiang Uyghur Autonomous Region, College of Chemistry and Chemical Engineering, Xinjiang University, Urumqi 830046, China b Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, China c Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
Chitosan coated MCNC materials were prepared by a simple one-pot method. The Fe3O4@CS MCNC materials showed super hydrophilicity. The material had high selectivity and efficiency for enrichment of glycopeptides.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 March 2014 Received in revised form 9 May 2014 Accepted 25 May 2014 Available online xxx
Selective enrichment of glycopeptides prior to the mass spectrometry (MS) analysis is essential due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among the enrichment approaches, hydrophilic interaction liquid chromatography (HILIC) based on magnetic separation has become a popular method in recent years. As the conventional synthesis procedures of these materials are tedious and time-consuming with at least four steps. Herein, magnetic colloidal nanocrystal clusters coated with chitosan (Fe3O4@CS MCNCs) have been successfully prepared by a simple one-pot method. The resulting Fe3O4@CS MCNCs demonstrated an excellent ability for glycopeptide enrichment with high selectivity, low detection limit and high binding capacity. Furthermore, in the analysis of real complicated biological sample, 283 unique N-glycosylation sites corresponding to 175 glycosylated proteins were identified in three replicate analyses of 45 mg protein sample extracted from HeLa cells, indicating the great potential in detection and identification of low abundant glycopeptides in glycoproteome analysis. ã 2014 Published by Elsevier B.V.
Keywords: Glycopeptides Magnetic colloidal nanocrystal clusters Chitosan Hydrophilic interaction liquid chromatography
* Corresponding author at: No. 14 Shengli Road, Urumqi, China. Tel.: +86 991 8582087; fax: +86 991 8582087. ** Corresponding author at: No. 457 Zhongshan Road, Dalian, China. Tel.: +86 411 84379610; fax: +86 411 84379620. E-mail addresses: [email protected] (S. Feng), [email protected] (H. Zou).
1. Introduction
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Protein glycosylation is one of the most important posttranslational modifications (PTMs) and plays important structural and functional roles in multiple biological processes, including cell –cell interaction, immune defence, cell growth and differentiation [1,2]. And approximately 50% of the mammalian proteome is composed of glycoproteins [3]. Aberrant glycosylation on some
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http://dx.doi.org/10.1016/j.aca.2014.05.037 0003-2670/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: C. Fang, et al., One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.037
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important proteins is highly associated with various types of diseases such as cancer and neuromuscular disorders [4,5]. Nglycosylated proteins are also important for disease prognosis, diagnosis, and therapeutic response to drugs [6,7]. Consequently, the analysis of protein glycosylation is of great significance not only for biomedical research but also for biotechnology industries [8,9]. Nowadays, mass spectrometry (MS) based techniques has emerged as the most important and powerful tool for the characterization of protein glycosylation. However, the high dynamic range of glycoproteins, inherent low abundance of glycopeptides and the severely ion suppression effect caused by the co-existence of nonglycosylated peptides make the direct MS analysis of glycoproteins/glycopeptides a challenge. Therefore, efficient extraction of glycoproteins/glycopeptides from highly complicated mixtures is absolutely necessary prior to MS analysis. Several methods including hydrophilic interaction liquid chromatography (HILIC), lectin affinity chromatography, hydrazide chemistry and boronic acid chemistry have been devoted to the enrichment of glycoproteins/glycopeptides [10–13]. Among them, the enrichment strategy based on HILIC has been well developed in recent years and gained increasing popularity due to the simple operating process, good reproducibility, no bias and avoids irreversible alteration of glycan composition [14]. In last decades, magnetic separation has become an effective isolation technique, which could achieve better separation compared with traditional approaches [15]. Combining HILIC-based strategy with magnetic separation technology was advisable by simultaneously taking both of the advantages and thus captures glycopeptides from complex biological sample in a rapid and efficient manner. Xiong et al. have prepared Fe3O4@SiO2@PEG-Maltose magnetic nanoparticles for selective glycopeptide enrichment. The hydrophilic maltose group grafted on the material dramatically increased due to PEG brushes on the surface and thus obtained high specifity, high sensitivity, and large binding capacity to glycopeptides [16]. Yeh et al. reported a novel bead based ZIC-HILIC synthesis method by spontaneous acid-catalyzed polymerization on iron oxide magnetic nanoparticles. Being applied for the enrichment of glycopeptides, the material presented high selectivity and recovery [17]. Notwithstanding these successful examples, the design, synthesis and applications of novel and more convenient materials are still attracting extensive attentions aimed at improving the glycoproteins/glycopeptides enrichment specificity and recovery. Normally, multifunctioned hydrophilic magnetic materials were prepared by at least four steps: firstly, synthesizing Fe3O4 magnetic nanoparticles (MNPs), then coating the MNPs with SiO2 shell followed by activating with -NH2, -Cl or other function groups, and finally grafting hydrophilic molecules. They were successful, however, the synthesis processes were tedious and time-consuming. Recently, Gao et al. have adopted one-step solvothermal method for the synthesis of magnetic colloidal nanocrystal clusters (MCNCs) [18]. Li et al. have reported a one-pot approach to obtain mesoporous magnetic colloidal nanocrystal clusters (MMCNCs) for drug delivery; the agarose acted as both the porogen and the stabilizer, and the large surface of agarosecovered MMCNCs can be further modified for drug carrying [19]. Chitosan is a kind of a polysaccharide which is mostly used in the biomedical domain for its biodegradability, biocompatibility and antibacterial activity [20,21]. Like other disaccharides or polysaccharides such as maltose, agarose and cellulose which have been employed for glycopeptide enrichment [22–24], the hydrophilicity introduced by its polar groups (-NH2, -OH) equipped chitosan with the ability to capture glycopeptides through interaction with the glycan moieties by hydrogen bonding [20,25,26]. Based on these, the synthesis of chitosan coated MCNCs by a convenient one-pot method for glycopeptide enrichment would be very attractive.
Herein, we prepared magnetic colloidal nanocrystal clusters coated with chitosan (Fe3O4@CS MCNCs) via one-pot solvothermal method for the enrichment of glycopeptides. The as-synthesized Fe3O4@CS MCNCs were proved to have a colloidal nanocrystal clusters structure and unique magnetic response. The outermost chitosan layer endowed MCNCs with high hydrophilic surface property, which not only enhanced the water dispersity of MCNCs, but also bonded functional groups for large binding capacity of glycopeptides. These composite MCNCs for the enrichment of glycopeptides were investigated by tryptic digests of both standard glycoprotein and complex biological samples. The experimental results indicated that the proposed procedure with a simple synthesized operation, high selectively and great capability in the selective enrichment of low-abundant glycopeptides in complicated biological samples.
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2. Experimental
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2.1. Materials
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Human serum immunoglobulin G (human IgG), trypsin (TPCK treated), dithiothreitol (DTT), iodoacetamide (IAA), chitosan (CS, low molecular weight), 2,5-dihydroxyl benzoic acid (DHB) and trifluoroacetic acid (TFA) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Iron(III) chloride hexahydrate (FeCl36H2O), ammonium acetate (NH4Ac) and ethylene glycol (EG) were obtained from Tianjin Chemical Plant (Tianjin, China). Formic acid (FA) was from Fluka (Buches, Germany). Acetonitrile (ACN, HPLC grade) was from Merck (Darmstadt, Germany). PNGase F was purchased from New England Biolabs, (Beverly, MA, USA). Pure water (18.4 MV cm) used in all experiments was purified by a Milli-Q system (Millipore, Milford, MA, USA).
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2.2. Synthesis of Fe3O4@CS MCNCs
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In a typical experiment, 2.163 g of FeCl36H2O was dissolved in EG (70 mL) by ultrasonication for 15 min to form a clear solution, followed by the addition of 3.7 g of NH4Ac and 1.0 g of CS. The mixture was heated at 160 C for 2 h under nitrogen atmosphere, then sealed in 100 mL of teflon-lined stainless-steel autoclave and heated at 200 C for 16 h. After the reaction, the mixture was cooled to room temperature and isolated by a magnet and washed with pure water and ethanol, respectively.
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2.3. Material characterization
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Transmission electron microscopy (TEM) image was obtained by JEOL JEM-2000 EX transmission electron microscope (JEOL, Tokyo, Japan). TGA data analyses were performed on Netzsch STA 409 PC thermal analysis system (NETZSCH, Selb, Germany) under air flow. Elemental analyses were performed on Vario EL III (Elementar, Hanau, Germany). Zeta (z) potential measurements were conducted on Nano-ZS90 instrument (Malvern, Worcestershire, United Kingdom) in water at 25 C. The saturation magnetization curve was carried out at room temperature on the Physical Property Measurement System 9 T (Quantum Design, San Diego, USA).
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2.4. Trypsin digestion of standard glycoprotein and protein mixture extracted from HeLa cells
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For standard glycoprotein preparation, human IgG was dissolved in 50 mM NH4HCO3 (pH 8.2) to a final concentration of 1 mg mL 1 and denatured by boiling for 10 min. The sample was reduced by 10 mM DTT at 60 C for 1 h and then alkylated by 20 mM IAA in the dark at room temperature for 30 min. Trypsin was added
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at the ratio of 1:25 (w/w) to digest proteins at 37 C overnight. The tryptic digests were stored at 20 C until further use. For protein mixture preparation, the HeLa cells were cultured in RPMI-1640 medium, supplemented with 10% bovine serum, 100 U mL 1 penicillin and streptomycin in a humidified atmosphere of 5% CO2 at 37 C. The cells were harvested at about 80% density, and the cell pellets were softly homogenized in an ice-cold lysis buffer containing 8 M urea, 50 mM Tris–HCl (pH 7.4), 65 mM DTT, 1% Triton X-100 (v/v), 1% protease cocktail (v/v), 1 mM EDGA, 1 mM EDTA, and 1 mM PMSF, sonicated for 400 W 120 s followed by centrifugation at 25,000 g for 1 h. Then the supernatant was precipitated with 5 volumes of ice-cold acetone/ethanol/acetic acid (v/v/v = 50/49.9/0.1) at 20 C overnight. The protein precipitant was centrifuged at 15,000 g for 30 min, and the pellet was washed with acetone and 75% ethanol, respectively. After being lyophilized to dryness, the HeLa cell proteins were redissolved in buffer containing 8 M urea and 100 mM NH4HCO3 (pH 8.2). The protein concentration was determined by Bradford assay, and the mixture was reduced by 10 mM DTT at 60 C for 1 h and then alkylated by 20 mM IAA in the dark at room temperature for 30 min followed by incubation with trypsin at the ratio of 1:25 (w/w) to digest proteins at 37 C overnight. After that, the protein concentration was diluted to 1 mg mL 1 with 100 mM NH4HCO3 buffer (pH 8.2) and stored at 20 C.
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2.5. Enrichment of glycopeptides with Fe3O4@CS MCNCs
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An amount of 500 mg Fe3O4@CS MCNCs were firstly washed and dispersed in 200 mL loading buffer (ACN/H2O/TFA = 88:11.5:0.5, v/v/v), Then 1 mg of human IgG tryptic peptides were mixed with the beads and incubated at room temperature for 30 min with gentle shaking. After that, the Fe3O4@CS MCNCs were isolated with a
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magnet, and the supernatant was removed, followed by washing with 200 mL loading buffer for three times. Then, the captured glycopeptides were eluted by 10 mL elution buffer (ACN/H2O/ TFA = 30:69.9:0.1, v/v/v) for 10 min at room temperature. Finally, the eluted fractions were directly analyzed or analyzed after deglycosylation by MALDI-TOF MS. For glycopeptide enrichment from complex sample, 150 mg tryptic digests of HeLa proteins were incubated with 1 mg Fe3O4@CS MCNCs in 400 mL loading buffer. The capture procedure was carried out under gentle shaking at room temperature for 30 min. Subsequently, the supernatant was removed, and the residues were rinsed with 400 mL of the loading buffer for four times. Finally, the trapped glycopeptides were eluted, lyophilized and then re-dissolved in 150 mL 10 mM NH4HCO3 (pH 7.5). 100 units of PNGase F was added to the solution and incubated at 37 C overnight to remove the glycan moieties for nano LC-MS/MS analysis.
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2.6. MS analysis
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Standard glycoprotein samples were analyzed by an AB Sciex 5800 MALDI-TOF mass spectrometer (AB Sciex, CA) equipped with a pulsed Nd/YAG laser at 355 nm in reflector positive ion mode. A 0.5 mL aliquot of the eluate and 0.5 mL of DHB matrix were sequentially dropped onto the MALDI plate for MS analysis. HeLa cell protein samples were analyzed by a LTQ Orbitrap Velos (Thermo, San Jose, CA) with an Accela 600 HPLC system (Thermo, San Jose, CA, USA) for separation. The delycosylated peptides were redissolved in FA/H2O (0.1:99.9, v/v) and loaded on a 4 cm reversed-phase trap column (200 mm i.d.) packed with C18 AQ beads (5 mm, 120 Å, Daison, Osaka, Japan), then separated using a homemade 1 cm C18 capillary analysis column (75 mm i.d.) packed
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Fig. 1. Characterizations of the synthesized Fe3O4@CS MCNCs. (a) Representative TEM image; (b) TGA curves of Fe3O4 and Fe3O4@CS MCNCs; (c) surface zeta (z)-potential of Fe3O4 and Fe3O4@CS MCNCs; and (d) magnetic hysteresis curve.
Please cite this article in press as: C. Fang, et al., One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.037
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with C18 AQ beads (3 mm, 120 Å, Daison, Osaka, Japan). The gradient elution was set as follows: from 0 to 5% buffer B (FA/ACN = 0.1:99.9, v/v) for 5 min, from 5 to 35% buffer B for 120 min, and from 35 to 80% buffer B for 30 min. After running with 80% buffer B for 10 min, the separation system was equilibrated by buffer A (FA/H2O = 0.1:99.9, v/v) for 15 min. The flow rate was adjusted to about 200 nL min 1, and the spray voltage was operated at 2.0 kV with the ion transfer capillary at 250 C. The MS/MS spectra were obtained in a data-dependent collision induced dissociation (CID) mode at 35.0% energy with one MS scan followed by 20 MS/MS scans, and the full MS was acquired from m/z 400 to 2000 with resolution 60,000.
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2.7. Database searching
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All acquired raw files were converted to *.mgf files using DTA Supercharge (v2.0a7). Then the *.mgf files were searched against the IPI human database (v3.80, including 86,719 entries, 2011, ftp://ftp.ebi.ac.uk/pub/databases/IPI). Cysteine carboxamidomethylation (+57.0215 Da) was set as a static modification; oxidation of methionine (+15.9949 Da) and a PNGase F catalyzed conversion of asparagines to aspartic acid at N-glycosites (+0.9840 Da) were set as the variable modifications. Mass tolerances were 10 ppm and 0.5 Da for the parent and fragment ions, respectively. A maximum of two missed cleavages was allowed, and the false positive rates (FDRs) were controlled to <1% for identification of peptides, proteins, and glycosylation sites.
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3. Results and discussion
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3.1. Preparation and characterization of Fe3O4@CS MCNCs
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Uniform Fe3O4@CS MCNCs were prepared by a simple modified hydrothermal method. Representative TEM image of the asprepared Fe3O4@CS MCNCs is shown in Fig. 1a, which demonstrated that spherical nanocrystal clusters with an average diameter of about 140 nm were formed. These sub-micrometer sized clusters were formed by the aggregation of many small nanocrystals with sizes of 15–20 nm; in this case, the primary nanocrystals nucleate first and then aggregate into larger secondary particles. The obtained Fe3O4@CS MCNCs were similar to those synthesized using other stabilizers [27–29]. TG analyses were employed to estimate the weight ratio of inorganic and organic components in Fe3O4@CS MCNCs. Fig. 1b showed that the weight loss of Fe3O4@CS MCNCs over 200 C is about 27.41%, together with the elemental analysis that the nitrogen content of the material was 1.958 wt%, and the surface zeta potential switched to 9.6 mV (Fig. 1c); all these results demonstrated the high amount of CS with the Fe3O4@CS MCNCs. What is more, as presented in Fig. 1d, the magnetic saturation (Ms) value of the material is 46.1 emu g 1, indicating that the material has good ability in rapid magnetic separation. There were several other reports for glycopeptide enrichment using HILIC based beads. Liang and co-workers prepared silica-based click maltose [23] and click chitooligosaccharide [26] for the
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Fig. 2. MALDI-TOF MS spectra of 300 fmol tryptic digest of human IgG. (a) Direct analysis (300 fmol), (b) analysis after enrichment using Fe3O4@CS MCNCs (equal to 15 fmol), (c) analysis after enrichment using Fe3O4 MNPs (equal to 15 fmol), (d) analysis of the deglycosylated peptides enriched by Fe3O4@CS MCNCs. Mannose ( ), galactose ( ), fucose ( ), and GlcNAc ( ).
Please cite this article in press as: C. Fang, et al., One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.037
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Fig. 3. MALDI-TOF MS spectra of tryptic digests of human IgG analysis after enrichment using Fe3O4@CS MCNCs. (a) 50 fmol, (b) 25 fmol, (c) 10 fmol, and (d) 8 fmol. * represent glycopeptides. 247
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enrichment of glycopeptides. The synthesized procedure could be done in a modular condition with very high reaction efficiency by click chemistry; however, the large amount of time needed and welldeveloped skills required made this approach less accessible. Kuo et al. utilized amine-derivatized Fe3O4 nanoparticles to enrich glycopeptides [30]. The low density and small amount of grafted amine group on the nanoparticles limited its selectivity and capacity for glycopeptides. Herein, the hydrophilic Fe3O4@CS MCNCs were prepared with high amount of chitosan and uniform magnetic property through one-pot method, which provided a readily and robust way to prepare novel materials for glycopeptide enrichment.
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3.2. Glycopeptide enrichment from standard glycoprotein
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In light of enrichment efficiency of the synthesized Fe3O4@CS MNPs, the applicability of the selective enrichment for
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Fig. 4. The amount of MCNCs influencing intensity of five selected glycopeptides from tryptic digests of human IgG (8 mg) after enrichment using Fe3O4@CS MCNCs.
261 glycopeptides was investigated using tryptic human IgG digests. 262 As shown in Fig. 2a, without any enrichment, the MS spectra were 263 dominated by non-glycopeptides, while the MS signals of 264 glycopeptides were suppressed severely, and only two glycopep265 tides were apparent with lower MS intensities and signal-to-noise 266 (S/N) ratios compared to non-glycopeptides. However, after the 267 enrichment with Fe3O4@CS MCNCs, the scenario were significantly Q11 268 improved and 21 glycopeptides (Table S1, see Supporting 269 Information) could be clearly detected with high MS intensities 270 and improved S/N ratios; meanwhile the high abundant of non271 glycosylated peptides were efficiently removed (Fig. 2b). For 272 comparison, bare Fe3O4 MNPs also tested for glycopeptide 273 enrichment; however, only four glycopeptides were detected with 274 lower S/N ratios and a large amount of non-glycopeptides still 275 dominated (Fig. 2c). In addition, after treated with PNGase F, the 276 eluted fraction from Fe3O4@CS MCNCs enrichment was analyzed 277 by MALDI-TOF MS (Fig. 2d). It can be seen that the signals of 278 glycopeptides disappeared, and only the deglycosylated peptides 279 were detected, which demonstrated that the peaks in Fig. 2b were 280 glycopeptides. The better efficiency obtained might be attributed 281 to the high amount of CS unit on Fe3O4@CS MCNCs which endows 282 the materials with good hydrophilic property. Owing to the 283 hydrophilicity difference between glycopeptides and non-glyco284 peptides, low abundant glycopeptides in the complex peptides' 285 mixtures were captured on the hydrophilic polysaccharide shells 286 through the multivalent hydrogen-bonding interaction and non287 glycopeptides could be moved through washing process [14,31,32]. 288 In the previous studies, Xiong et al. successfully fabricated 289 Fe3O4@SiO2@PEG-Maltose [16] and Fe3O4@SiO2@(HA/CS)10 [25] 290 nanoparticles, which exhibited excellent enrichment perform291 ances and identified 27 and 24 glycopeptides from tryptic human 292 IgG digests, respectively. In this work, a total of 21 glycopeptides were determined from tryptic human IgG digests, which was a Q12 293 294 little less than those derived from Fe3O4@SiO2@PEG-Maltose 295 and Fe3O4@SiO2@(HA/CS)10 nanoparticles. It should be empha296 sized that the time required for the preparation of the above 297 mentioned nanoparticles was more than 100 h, which was over
Please cite this article in press as: C. Fang, et al., One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.037
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five times than ours. Based on this, one-pot method showed significant advantages in time-saving and steps eliminating; meanwhile the resulted Fe3O4@CS MCNCs material also can keep sufficient selectivity and specificity. Detection limit is a key criterion to evaluate the enrichment efficiency of nanomaterials. Herein, tryptic human IgG digests (50 fmol, 25 fmol, 10 fmol and 8 fmol) were used to evaluate the detection limit. As presented in Fig. 3, four glycopeptides still could be well detected after the enrichment by Fe3O4@CS MCNCs even though the amount of glycopeptides was as low as 8 fmol. Among the four glycopeptide peaks, the lowest S/N ratio was 22.4 (the peak of m/z 2968.9), which meant that the detection limit was lower than 8 fmol. The results indicated that Fe3O4@CS MCNCs presented the same excellent performance with zwitterionic polymer brushes hybrid silica nanoparticles (10 fmol) [33] and much higher sensitivity than that of silica-based click maltose (30 fmol) [34]. The binding capacity of Fe3O4@CS MCNCs toward glycopeptides was also investigated according to Xiong et al.'s report [25]. Different amount of MNPs were added to a fixed amount of tryptic digests of human IgG (8 mg). After the enrichment, the eluate (0.5 mL from 10 mL total) was analyzed with MALDI-TOF MS. As shown in Fig. 4, when the amount of material added was 500 mg, the intensity of five selected glycopeptides reached the maximum and almost remained the same even when the amount of material increased. According to this method, the binding capacity of Fe3O4@CS MCNCs was calculated to be about 17.5 mg g 1.
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3.3. Glycopeptide enrichment from HeLa cell proteins
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Encouraged by the excellent efficiency of glycopeptide enrichment in standard glycoprotein digests, the Fe3O4@CS MCNCs were further applied for analysis of glycoproteome of complex protein mixture extracted from HeLa cells. 45 mg tryptic digests of HeLa cell proteins was analyzed as described above. After controlling the identification FDR <1%, a total of 283 unique N-glycosylation sites in 273 unique glycopeptides, corresponding to 175 glycoproteins were identified (with N-X-T/S motif, X can be any amino acid except proline) in three replicate analyses. The information of the identified glycopeptides, N-glycosylation sites and glycoproteins are given in Table S2 (see Supporting Information). The results clearly indicated that Fe3O4@CS MCNCs were capable of highly selective trapping glycopeptides for glycosylation characterization of glycoproteins in real biological samples.
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4. Conclusion
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In conclusion, Fe3O4@CS MCNCs have been successfully prepared by an easily accessible one-pot solvothermal strategy. The as-prepared materials carry forward the virtues of being easy-to-prepare, being cost-effective, and being readily separated and dispersed. Due to the high amount of coated CS, good performance of glycopeptide enrichment with high selectivity, low detection limit and high binding capacity could be obtained by using Fe3O4@CS MCNCs. Moreover, the prepared material presented great potential in detection and identification with low abundant glycopeptides in minutes of biological samples. Additionally, it could be extended to the design of other functional magnetic based materials with colloidal nanocrystal clusters structures by one-pot method for drug delivery system, biological sensor, and so on.
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Acknowledgements
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This work were supported by China State Key Basic Research Program Grant (2013CB911202), Creative Research Group Project
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of NSFC (21321064), National Natural Sciences Foundation of China (21075105 and 21165017), and Educational Commission of the Xinjiang Uygur Autonomous Region of China (XJEDU2010I0). The funders have no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.05.037.
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References
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[1] K. Ohtsubo, J.D. Marth, Glycosylation in cellular mechanisms of health and disease, Cell 126 (2006) 855–867. [2] A. Helenius, M. Aebi, Intracellular functions of N-linked glycans, Science 291 (2001) 2364–2369. [3] R. Apweiler, H. Hermjakob, N. Sharon, On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database, Biochim. Biophys. Acta 1473 (1999) 4–8. [4] R.J. Woods, C.J. Edge, R.A. Dwek, Protein surface oligosaccharides and protein function, Nat. Struct. Mol. Biol. 1 (1994) 499–501. [5] P.T. Martin, H.H. Freeze, Glycobiology of neuromuscular disorders, Glycobiology 13 (2003) 67R–75R. [6] M.L. Montpetit, P.J. Stocker, T.A. Schwetz, J.M. Harper, S.A. Norring, L. Schaffer, S.J. North, J. Jang-Lee, T. Gilmartin, S.R. Head, Regulated and aberrant glycosylation modulate cardiac electrical signaling, Proc. Natl. Acad. Sci. 106 (2009) 16517–16522. [7] J.A. Ludwig, J.N. Weinstein, Biomarkers in cancer staging, prognosis and treatment selection, Nat. Rev. Cancer 5 (2005) 845–856. [8] D.F. Zielinska, F. Gnad, J.R. Wisniewski, M. Mann, Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints, Cell 141 (2010) 897–907. [9] I.M. Lazar, A.C. Lazar, D.F. Cortes, J.L. Kabulski, Recent advances in the MS analysis of glycoproteins: theoretical considerations, Electrophoresis 32 (2011) 3–13. [10] C.-Y. Lin, Y.-C. Ma, P.-J. Pai, G.-R. Her, A comparative study of glycoprotein concentration, glycoform profile and glycosylation site occupancy using isotope labeling and electrospray linear ion trap mass spectrometry, Anal. Chim. Acta 728 (2012) 49–56. [11] S. Feng, N. Yang, S. Pennathur, S. Goodison, D.M. Lubman, Enrichment of glycoproteins using nanoscale chelating concanavalin A monolithic capillary chromatography, Anal. Chem. 81 (2009) 3776–3783. [12] H. Zhang, X. Li, D.B. Martin, R. Aebersold, Identification and quantification of Nlinked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry, Nat. Biotechnol. 21 (2003) 660–666. [13] L. Liu, Y. Zhang, L. Zhang, G. Yan, J. Yao, P. Yang, H. Lu, Highly specific revelation of rat serum glycopeptidome by boronic acid-functionalized mesoporous silica, Anal. Chim. Acta 753 (2012) 64–72. [14] P.J. Boersema, S. Mohammed, A.J. Heck, Hydrophilic interaction liquid chromatography (HILIC) in proteomics, Anal. Bioanal. Chem. 391 (2008) 151–159. [15] L.H. Reddy, J.L. Arias, J. Nicolas, P. Couvreur, Magnetic nanoparticles: design and characterization, toxicity and biocompatibility, pharmaceutical and biomedical applications, Chem. Rev. 112 (2012) 5818–5878. [16] Z. Xiong, L. Zhao, F. Wang, J. Zhu, H. Qin, R. a. Wu, W. Zhang, H. Zou, Synthesis of branched PEG brushes hybrid hydrophilic magnetic nanoparticles for the selective enrichment of N-linked glycopeptides, Chem. Commun. 48 (2012) 8138–8140. [17] C.H. Yeh, S.H. Chen, D.T. Li, H.P. Lin, H.J. Huang, C.I. Chang, W.L. Shih, C.L. Chern, F.K. Shi, J.L. Hsu, Magnetic bead-based hydrophilic interaction liquid chromatography for glycopeptide enrichments, J. Chromatogr. A 1224 (2012) 70–78. [18] J. Gao, X. Ran, C. Shi, H. Cheng, T. Cheng, Y. Su, One-step solvothermal synthesis of highly water-soluble, negatively charged superparamagnetic Fe3O4 colloidal nanocrystal clusters, Nanoscale 5 (2013) 7026–7033. [19] D. Li, J. Tang, C. Wei, J. Guo, S. Wang, D. Chaudhary, C. Wang, Doxorubicinconjugated mesoporous magnetic colloidal nanocrystal clusters stabilized by polysaccharide as a smart anticancer drug vehicle, Small 8 (2012) 2690–2697. [20] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 31 (2006) 603–632. [21] J.-K. Francis Suh, H.W. Matthew, Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review, Biomaterials 21 (2000) 2589–2598. [22] Y. Wada, M. Tajiri, S. Yoshida, Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics, Anal. Chem. 76 (2004) 6560–6565. [23] L. Yu, X. Li, Z. Guo, X. Zhang, X. Liang, Hydrophilic interaction chromatography based enrichment of glycopeptides by using click maltose: a matrix with high selectivity and glycosylation heterogeneity coverage, Chem. Eur. J. 15 (2009) 12618–12626.
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[24] M.H. Selman, M. Hemayatkar, A.M. Deelder, M. Wuhrer, Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides, Anal. Chem. 83 (2011) 2492–2499. [25] Z. Xiong, H. Qin, H. Wan, G. Huang, Z. Zhang, J. Dong, L. Zhang, W. Zhang, H. Zou, Layer-by-layer assembly of multilayer polysaccharide coated magnetic nanoparticles for the selective enrichment of glycopeptides, Chem. Commun. 49 (2013) 9284–9286. [26] H. Huang, Y. Jin, M. Xue, L. Yu, Q. Fu, Y. Ke, C. Chu, X. Liang, A novel click chitooligosaccharide for hydrophilic interaction liquid chromatography, Chem. Commun. 45 (2009) 6973–6975. [27] J. Ge, Y. Hu, M. Biasini, W.P. Beyermann, Y. Yin, Superparamagnetic magnetite colloidal nanocrystal clusters, Angew. Chem. Int. Ed. 46 (2007) 4342–4345. [28] B. Luo, S. Xu, A. Luo, W.-R. Wang, S.-L. Wang, J. Guo, Y. Lin, D.-Y. Zhao, C.-C. Wang, Mesoporous biocompatible and acid-degradable magnetic colloidal nanocrystal clusters with sustainable stability and high hydrophobic drug loading capacity, ACS Nano 5 (2011) 1428–1435. [29] C. Cheng, Y. Wen, X. Xu, H. Gu, Tunable synthesis of carboxyl-functionalized magnetite nanocrystal clusters with uniform size, J. Mater. Chem. 19 (2009) 8782–8788.
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[30] C. Kuo, I. Wu, H. Hsiao, K. Khoo, Rapid glycopeptide enrichment and Nglycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles, Anal. Bioanal. Chem. 402 (2012) 2765–2776. [31] M. Thaysen-Andersen, I.B. Thøgersen, H.J. Nielsen, U. Lademann, N. Brünner, J.J. Enghild, P. Højrup, Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1, Mol. Cell. Proteomics 6 (2007) 638–647. [32] P. Hägglund, J. Bunkenborg, F. Elortza, O.N. Jensen, P. Roepstorff, A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation, J. Proteome. Res. 3 (2004) 556–566. [33] G. Huang, Z. Xiong, H. Qin, J. Zhu, Z. Sun, Y. Zhang, X. Peng, H. Zou, Synthesis of zwitterionic polymer brushes hybrid silica nanoparticles via controlled polymerization for highly efficient enrichment of glycopeptides, Anal. Chim. Acta 809 (2014) 61–68. [34] J. Zhu, F. Wang, R. Chen, K. Cheng, B. Xu, Z. Guo, X. Liang, M. Ye, H. Zou, Centrifugation assisted microreactor enables facile integration of trypsin digestion, hydrophilic interaction chromatography enrichment, and oncolumn deglycosylation for rapid and sensitive N-glycoproteome analysis, Anal. Chem. 84 (2012) 5146–5153.
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One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides Chunli Fang a , Zhichao Xiong b , Hongqiang Qin c , Guang Huang c, Jing Liu c , Mingliang Ye c, Shun Feng a, * , Hanfa Zou c, ** a Key Laboratory of Oil Gas Fine Chemicals, Ministry of Education Xinjiang Uyghur Autonomous Region, College of Chemistry and Chemical Engineering, Xinjiang University, Urumqi 830046, China b Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, China c Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
Chitosan coated MCNC materials were prepared by a simple one-pot method. The Fe3O4@CS MCNC materials showed super hydrophilicity. The material had high selectivity and efficiency for enrichment of glycopeptides.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 March 2014 Received in revised form 9 May 2014 Accepted 25 May 2014 Available online xxx
Selective enrichment of glycopeptides prior to the mass spectrometry (MS) analysis is essential due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among the enrichment approaches, hydrophilic interaction liquid chromatography (HILIC) based on magnetic separation has become a popular method in recent years. As the conventional synthesis procedures of these materials are tedious and time-consuming with at least four steps. Herein, magnetic colloidal nanocrystal clusters coated with chitosan (Fe3O4@CS MCNCs) have been successfully prepared by a simple one-pot method. The resulting Fe3O4@CS MCNCs demonstrated an excellent ability for glycopeptide enrichment with high selectivity, low detection limit and high binding capacity. Furthermore, in the analysis of real complicated biological sample, 283 unique N-glycosylation sites corresponding to 175 glycosylated proteins were identified in three replicate analyses of 45 mg protein sample extracted from HeLa cells, indicating the great potential in detection and identification of low abundant glycopeptides in glycoproteome analysis. ã 2014 Published by Elsevier B.V.
Keywords: Glycopeptides Magnetic colloidal nanocrystal clusters Chitosan Hydrophilic interaction liquid chromatography
* Corresponding author at: No. 14 Shengli Road, Urumqi, China. Tel.: +86 991 8582087; fax: +86 991 8582087. ** Corresponding author at: No. 457 Zhongshan Road, Dalian, China. Tel.: +86 411 84379610; fax: +86 411 84379620. E-mail addresses: [email protected] (S. Feng), [email protected] (H. Zou).
1. Introduction
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Protein glycosylation is one of the most important posttranslational modifications (PTMs) and plays important structural and functional roles in multiple biological processes, including cell –cell interaction, immune defence, cell growth and differentiation [1,2]. And approximately 50% of the mammalian proteome is composed of glycoproteins [3]. Aberrant glycosylation on some
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http://dx.doi.org/10.1016/j.aca.2014.05.037 0003-2670/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: C. Fang, et al., One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.037
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important proteins is highly associated with various types of diseases such as cancer and neuromuscular disorders [4,5]. Nglycosylated proteins are also important for disease prognosis, diagnosis, and therapeutic response to drugs [6,7]. Consequently, the analysis of protein glycosylation is of great significance not only for biomedical research but also for biotechnology industries [8,9]. Nowadays, mass spectrometry (MS) based techniques has emerged as the most important and powerful tool for the characterization of protein glycosylation. However, the high dynamic range of glycoproteins, inherent low abundance of glycopeptides and the severely ion suppression effect caused by the co-existence of nonglycosylated peptides make the direct MS analysis of glycoproteins/glycopeptides a challenge. Therefore, efficient extraction of glycoproteins/glycopeptides from highly complicated mixtures is absolutely necessary prior to MS analysis. Several methods including hydrophilic interaction liquid chromatography (HILIC), lectin affinity chromatography, hydrazide chemistry and boronic acid chemistry have been devoted to the enrichment of glycoproteins/glycopeptides [10–13]. Among them, the enrichment strategy based on HILIC has been well developed in recent years and gained increasing popularity due to the simple operating process, good reproducibility, no bias and avoids irreversible alteration of glycan composition [14]. In last decades, magnetic separation has become an effective isolation technique, which could achieve better separation compared with traditional approaches [15]. Combining HILIC-based strategy with magnetic separation technology was advisable by simultaneously taking both of the advantages and thus captures glycopeptides from complex biological sample in a rapid and efficient manner. Xiong et al. have prepared Fe3O4@SiO2@PEG-Maltose magnetic nanoparticles for selective glycopeptide enrichment. The hydrophilic maltose group grafted on the material dramatically increased due to PEG brushes on the surface and thus obtained high specifity, high sensitivity, and large binding capacity to glycopeptides [16]. Yeh et al. reported a novel bead based ZIC-HILIC synthesis method by spontaneous acid-catalyzed polymerization on iron oxide magnetic nanoparticles. Being applied for the enrichment of glycopeptides, the material presented high selectivity and recovery [17]. Notwithstanding these successful examples, the design, synthesis and applications of novel and more convenient materials are still attracting extensive attentions aimed at improving the glycoproteins/glycopeptides enrichment specificity and recovery. Normally, multifunctioned hydrophilic magnetic materials were prepared by at least four steps: firstly, synthesizing Fe3O4 magnetic nanoparticles (MNPs), then coating the MNPs with SiO2 shell followed by activating with -NH2, -Cl or other function groups, and finally grafting hydrophilic molecules. They were successful, however, the synthesis processes were tedious and time-consuming. Recently, Gao et al. have adopted one-step solvothermal method for the synthesis of magnetic colloidal nanocrystal clusters (MCNCs) [18]. Li et al. have reported a one-pot approach to obtain mesoporous magnetic colloidal nanocrystal clusters (MMCNCs) for drug delivery; the agarose acted as both the porogen and the stabilizer, and the large surface of agarosecovered MMCNCs can be further modified for drug carrying [19]. Chitosan is a kind of a polysaccharide which is mostly used in the biomedical domain for its biodegradability, biocompatibility and antibacterial activity [20,21]. Like other disaccharides or polysaccharides such as maltose, agarose and cellulose which have been employed for glycopeptide enrichment [22–24], the hydrophilicity introduced by its polar groups (-NH2, -OH) equipped chitosan with the ability to capture glycopeptides through interaction with the glycan moieties by hydrogen bonding [20,25,26]. Based on these, the synthesis of chitosan coated MCNCs by a convenient one-pot method for glycopeptide enrichment would be very attractive.
Herein, we prepared magnetic colloidal nanocrystal clusters coated with chitosan (Fe3O4@CS MCNCs) via one-pot solvothermal method for the enrichment of glycopeptides. The as-synthesized Fe3O4@CS MCNCs were proved to have a colloidal nanocrystal clusters structure and unique magnetic response. The outermost chitosan layer endowed MCNCs with high hydrophilic surface property, which not only enhanced the water dispersity of MCNCs, but also bonded functional groups for large binding capacity of glycopeptides. These composite MCNCs for the enrichment of glycopeptides were investigated by tryptic digests of both standard glycoprotein and complex biological samples. The experimental results indicated that the proposed procedure with a simple synthesized operation, high selectively and great capability in the selective enrichment of low-abundant glycopeptides in complicated biological samples.
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2. Experimental
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2.1. Materials
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Human serum immunoglobulin G (human IgG), trypsin (TPCK treated), dithiothreitol (DTT), iodoacetamide (IAA), chitosan (CS, low molecular weight), 2,5-dihydroxyl benzoic acid (DHB) and trifluoroacetic acid (TFA) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Iron(III) chloride hexahydrate (FeCl36H2O), ammonium acetate (NH4Ac) and ethylene glycol (EG) were obtained from Tianjin Chemical Plant (Tianjin, China). Formic acid (FA) was from Fluka (Buches, Germany). Acetonitrile (ACN, HPLC grade) was from Merck (Darmstadt, Germany). PNGase F was purchased from New England Biolabs, (Beverly, MA, USA). Pure water (18.4 MV cm) used in all experiments was purified by a Milli-Q system (Millipore, Milford, MA, USA).
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2.2. Synthesis of Fe3O4@CS MCNCs
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In a typical experiment, 2.163 g of FeCl36H2O was dissolved in EG (70 mL) by ultrasonication for 15 min to form a clear solution, followed by the addition of 3.7 g of NH4Ac and 1.0 g of CS. The mixture was heated at 160 C for 2 h under nitrogen atmosphere, then sealed in 100 mL of teflon-lined stainless-steel autoclave and heated at 200 C for 16 h. After the reaction, the mixture was cooled to room temperature and isolated by a magnet and washed with pure water and ethanol, respectively.
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2.3. Material characterization
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Transmission electron microscopy (TEM) image was obtained by JEOL JEM-2000 EX transmission electron microscope (JEOL, Tokyo, Japan). TGA data analyses were performed on Netzsch STA 409 PC thermal analysis system (NETZSCH, Selb, Germany) under air flow. Elemental analyses were performed on Vario EL III (Elementar, Hanau, Germany). Zeta (z) potential measurements were conducted on Nano-ZS90 instrument (Malvern, Worcestershire, United Kingdom) in water at 25 C. The saturation magnetization curve was carried out at room temperature on the Physical Property Measurement System 9 T (Quantum Design, San Diego, USA).
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2.4. Trypsin digestion of standard glycoprotein and protein mixture extracted from HeLa cells
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For standard glycoprotein preparation, human IgG was dissolved in 50 mM NH4HCO3 (pH 8.2) to a final concentration of 1 mg mL 1 and denatured by boiling for 10 min. The sample was reduced by 10 mM DTT at 60 C for 1 h and then alkylated by 20 mM IAA in the dark at room temperature for 30 min. Trypsin was added
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at the ratio of 1:25 (w/w) to digest proteins at 37 C overnight. The tryptic digests were stored at 20 C until further use. For protein mixture preparation, the HeLa cells were cultured in RPMI-1640 medium, supplemented with 10% bovine serum, 100 U mL 1 penicillin and streptomycin in a humidified atmosphere of 5% CO2 at 37 C. The cells were harvested at about 80% density, and the cell pellets were softly homogenized in an ice-cold lysis buffer containing 8 M urea, 50 mM Tris–HCl (pH 7.4), 65 mM DTT, 1% Triton X-100 (v/v), 1% protease cocktail (v/v), 1 mM EDGA, 1 mM EDTA, and 1 mM PMSF, sonicated for 400 W 120 s followed by centrifugation at 25,000 g for 1 h. Then the supernatant was precipitated with 5 volumes of ice-cold acetone/ethanol/acetic acid (v/v/v = 50/49.9/0.1) at 20 C overnight. The protein precipitant was centrifuged at 15,000 g for 30 min, and the pellet was washed with acetone and 75% ethanol, respectively. After being lyophilized to dryness, the HeLa cell proteins were redissolved in buffer containing 8 M urea and 100 mM NH4HCO3 (pH 8.2). The protein concentration was determined by Bradford assay, and the mixture was reduced by 10 mM DTT at 60 C for 1 h and then alkylated by 20 mM IAA in the dark at room temperature for 30 min followed by incubation with trypsin at the ratio of 1:25 (w/w) to digest proteins at 37 C overnight. After that, the protein concentration was diluted to 1 mg mL 1 with 100 mM NH4HCO3 buffer (pH 8.2) and stored at 20 C.
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2.5. Enrichment of glycopeptides with Fe3O4@CS MCNCs
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An amount of 500 mg Fe3O4@CS MCNCs were firstly washed and dispersed in 200 mL loading buffer (ACN/H2O/TFA = 88:11.5:0.5, v/v/v), Then 1 mg of human IgG tryptic peptides were mixed with the beads and incubated at room temperature for 30 min with gentle shaking. After that, the Fe3O4@CS MCNCs were isolated with a
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magnet, and the supernatant was removed, followed by washing with 200 mL loading buffer for three times. Then, the captured glycopeptides were eluted by 10 mL elution buffer (ACN/H2O/ TFA = 30:69.9:0.1, v/v/v) for 10 min at room temperature. Finally, the eluted fractions were directly analyzed or analyzed after deglycosylation by MALDI-TOF MS. For glycopeptide enrichment from complex sample, 150 mg tryptic digests of HeLa proteins were incubated with 1 mg Fe3O4@CS MCNCs in 400 mL loading buffer. The capture procedure was carried out under gentle shaking at room temperature for 30 min. Subsequently, the supernatant was removed, and the residues were rinsed with 400 mL of the loading buffer for four times. Finally, the trapped glycopeptides were eluted, lyophilized and then re-dissolved in 150 mL 10 mM NH4HCO3 (pH 7.5). 100 units of PNGase F was added to the solution and incubated at 37 C overnight to remove the glycan moieties for nano LC-MS/MS analysis.
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2.6. MS analysis
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Standard glycoprotein samples were analyzed by an AB Sciex 5800 MALDI-TOF mass spectrometer (AB Sciex, CA) equipped with a pulsed Nd/YAG laser at 355 nm in reflector positive ion mode. A 0.5 mL aliquot of the eluate and 0.5 mL of DHB matrix were sequentially dropped onto the MALDI plate for MS analysis. HeLa cell protein samples were analyzed by a LTQ Orbitrap Velos (Thermo, San Jose, CA) with an Accela 600 HPLC system (Thermo, San Jose, CA, USA) for separation. The delycosylated peptides were redissolved in FA/H2O (0.1:99.9, v/v) and loaded on a 4 cm reversed-phase trap column (200 mm i.d.) packed with C18 AQ beads (5 mm, 120 Å, Daison, Osaka, Japan), then separated using a homemade 1 cm C18 capillary analysis column (75 mm i.d.) packed
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Fig. 1. Characterizations of the synthesized Fe3O4@CS MCNCs. (a) Representative TEM image; (b) TGA curves of Fe3O4 and Fe3O4@CS MCNCs; (c) surface zeta (z)-potential of Fe3O4 and Fe3O4@CS MCNCs; and (d) magnetic hysteresis curve.
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with C18 AQ beads (3 mm, 120 Å, Daison, Osaka, Japan). The gradient elution was set as follows: from 0 to 5% buffer B (FA/ACN = 0.1:99.9, v/v) for 5 min, from 5 to 35% buffer B for 120 min, and from 35 to 80% buffer B for 30 min. After running with 80% buffer B for 10 min, the separation system was equilibrated by buffer A (FA/H2O = 0.1:99.9, v/v) for 15 min. The flow rate was adjusted to about 200 nL min 1, and the spray voltage was operated at 2.0 kV with the ion transfer capillary at 250 C. The MS/MS spectra were obtained in a data-dependent collision induced dissociation (CID) mode at 35.0% energy with one MS scan followed by 20 MS/MS scans, and the full MS was acquired from m/z 400 to 2000 with resolution 60,000.
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2.7. Database searching
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All acquired raw files were converted to *.mgf files using DTA Supercharge (v2.0a7). Then the *.mgf files were searched against the IPI human database (v3.80, including 86,719 entries, 2011, ftp://ftp.ebi.ac.uk/pub/databases/IPI). Cysteine carboxamidomethylation (+57.0215 Da) was set as a static modification; oxidation of methionine (+15.9949 Da) and a PNGase F catalyzed conversion of asparagines to aspartic acid at N-glycosites (+0.9840 Da) were set as the variable modifications. Mass tolerances were 10 ppm and 0.5 Da for the parent and fragment ions, respectively. A maximum of two missed cleavages was allowed, and the false positive rates (FDRs) were controlled to <1% for identification of peptides, proteins, and glycosylation sites.
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3. Results and discussion
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3.1. Preparation and characterization of Fe3O4@CS MCNCs
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Uniform Fe3O4@CS MCNCs were prepared by a simple modified hydrothermal method. Representative TEM image of the asprepared Fe3O4@CS MCNCs is shown in Fig. 1a, which demonstrated that spherical nanocrystal clusters with an average diameter of about 140 nm were formed. These sub-micrometer sized clusters were formed by the aggregation of many small nanocrystals with sizes of 15–20 nm; in this case, the primary nanocrystals nucleate first and then aggregate into larger secondary particles. The obtained Fe3O4@CS MCNCs were similar to those synthesized using other stabilizers [27–29]. TG analyses were employed to estimate the weight ratio of inorganic and organic components in Fe3O4@CS MCNCs. Fig. 1b showed that the weight loss of Fe3O4@CS MCNCs over 200 C is about 27.41%, together with the elemental analysis that the nitrogen content of the material was 1.958 wt%, and the surface zeta potential switched to 9.6 mV (Fig. 1c); all these results demonstrated the high amount of CS with the Fe3O4@CS MCNCs. What is more, as presented in Fig. 1d, the magnetic saturation (Ms) value of the material is 46.1 emu g 1, indicating that the material has good ability in rapid magnetic separation. There were several other reports for glycopeptide enrichment using HILIC based beads. Liang and co-workers prepared silica-based click maltose [23] and click chitooligosaccharide [26] for the
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Fig. 2. MALDI-TOF MS spectra of 300 fmol tryptic digest of human IgG. (a) Direct analysis (300 fmol), (b) analysis after enrichment using Fe3O4@CS MCNCs (equal to 15 fmol), (c) analysis after enrichment using Fe3O4 MNPs (equal to 15 fmol), (d) analysis of the deglycosylated peptides enriched by Fe3O4@CS MCNCs. Mannose ( ), galactose ( ), fucose ( ), and GlcNAc ( ).
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Fig. 3. MALDI-TOF MS spectra of tryptic digests of human IgG analysis after enrichment using Fe3O4@CS MCNCs. (a) 50 fmol, (b) 25 fmol, (c) 10 fmol, and (d) 8 fmol. * represent glycopeptides. 247
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enrichment of glycopeptides. The synthesized procedure could be done in a modular condition with very high reaction efficiency by click chemistry; however, the large amount of time needed and welldeveloped skills required made this approach less accessible. Kuo et al. utilized amine-derivatized Fe3O4 nanoparticles to enrich glycopeptides [30]. The low density and small amount of grafted amine group on the nanoparticles limited its selectivity and capacity for glycopeptides. Herein, the hydrophilic Fe3O4@CS MCNCs were prepared with high amount of chitosan and uniform magnetic property through one-pot method, which provided a readily and robust way to prepare novel materials for glycopeptide enrichment.
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In light of enrichment efficiency of the synthesized Fe3O4@CS MNPs, the applicability of the selective enrichment for
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Fig. 4. The amount of MCNCs influencing intensity of five selected glycopeptides from tryptic digests of human IgG (8 mg) after enrichment using Fe3O4@CS MCNCs.
261 glycopeptides was investigated using tryptic human IgG digests. 262 As shown in Fig. 2a, without any enrichment, the MS spectra were 263 dominated by non-glycopeptides, while the MS signals of 264 glycopeptides were suppressed severely, and only two glycopep265 tides were apparent with lower MS intensities and signal-to-noise 266 (S/N) ratios compared to non-glycopeptides. However, after the 267 enrichment with Fe3O4@CS MCNCs, the scenario were significantly Q11 268 improved and 21 glycopeptides (Table S1, see Supporting 269 Information) could be clearly detected with high MS intensities 270 and improved S/N ratios; meanwhile the high abundant of non271 glycosylated peptides were efficiently removed (Fig. 2b). For 272 comparison, bare Fe3O4 MNPs also tested for glycopeptide 273 enrichment; however, only four glycopeptides were detected with 274 lower S/N ratios and a large amount of non-glycopeptides still 275 dominated (Fig. 2c). In addition, after treated with PNGase F, the 276 eluted fraction from Fe3O4@CS MCNCs enrichment was analyzed 277 by MALDI-TOF MS (Fig. 2d). It can be seen that the signals of 278 glycopeptides disappeared, and only the deglycosylated peptides 279 were detected, which demonstrated that the peaks in Fig. 2b were 280 glycopeptides. The better efficiency obtained might be attributed 281 to the high amount of CS unit on Fe3O4@CS MCNCs which endows 282 the materials with good hydrophilic property. Owing to the 283 hydrophilicity difference between glycopeptides and non-glyco284 peptides, low abundant glycopeptides in the complex peptides' 285 mixtures were captured on the hydrophilic polysaccharide shells 286 through the multivalent hydrogen-bonding interaction and non287 glycopeptides could be moved through washing process [14,31,32]. 288 In the previous studies, Xiong et al. successfully fabricated 289 Fe3O4@SiO2@PEG-Maltose [16] and Fe3O4@SiO2@(HA/CS)10 [25] 290 nanoparticles, which exhibited excellent enrichment perform291 ances and identified 27 and 24 glycopeptides from tryptic human 292 IgG digests, respectively. In this work, a total of 21 glycopeptides were determined from tryptic human IgG digests, which was a Q12 293 294 little less than those derived from Fe3O4@SiO2@PEG-Maltose 295 and Fe3O4@SiO2@(HA/CS)10 nanoparticles. It should be empha296 sized that the time required for the preparation of the above 297 mentioned nanoparticles was more than 100 h, which was over
Please cite this article in press as: C. Fang, et al., One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.037
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five times than ours. Based on this, one-pot method showed significant advantages in time-saving and steps eliminating; meanwhile the resulted Fe3O4@CS MCNCs material also can keep sufficient selectivity and specificity. Detection limit is a key criterion to evaluate the enrichment efficiency of nanomaterials. Herein, tryptic human IgG digests (50 fmol, 25 fmol, 10 fmol and 8 fmol) were used to evaluate the detection limit. As presented in Fig. 3, four glycopeptides still could be well detected after the enrichment by Fe3O4@CS MCNCs even though the amount of glycopeptides was as low as 8 fmol. Among the four glycopeptide peaks, the lowest S/N ratio was 22.4 (the peak of m/z 2968.9), which meant that the detection limit was lower than 8 fmol. The results indicated that Fe3O4@CS MCNCs presented the same excellent performance with zwitterionic polymer brushes hybrid silica nanoparticles (10 fmol) [33] and much higher sensitivity than that of silica-based click maltose (30 fmol) [34]. The binding capacity of Fe3O4@CS MCNCs toward glycopeptides was also investigated according to Xiong et al.'s report [25]. Different amount of MNPs were added to a fixed amount of tryptic digests of human IgG (8 mg). After the enrichment, the eluate (0.5 mL from 10 mL total) was analyzed with MALDI-TOF MS. As shown in Fig. 4, when the amount of material added was 500 mg, the intensity of five selected glycopeptides reached the maximum and almost remained the same even when the amount of material increased. According to this method, the binding capacity of Fe3O4@CS MCNCs was calculated to be about 17.5 mg g 1.
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3.3. Glycopeptide enrichment from HeLa cell proteins
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Encouraged by the excellent efficiency of glycopeptide enrichment in standard glycoprotein digests, the Fe3O4@CS MCNCs were further applied for analysis of glycoproteome of complex protein mixture extracted from HeLa cells. 45 mg tryptic digests of HeLa cell proteins was analyzed as described above. After controlling the identification FDR <1%, a total of 283 unique N-glycosylation sites in 273 unique glycopeptides, corresponding to 175 glycoproteins were identified (with N-X-T/S motif, X can be any amino acid except proline) in three replicate analyses. The information of the identified glycopeptides, N-glycosylation sites and glycoproteins are given in Table S2 (see Supporting Information). The results clearly indicated that Fe3O4@CS MCNCs were capable of highly selective trapping glycopeptides for glycosylation characterization of glycoproteins in real biological samples.
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4. Conclusion
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In conclusion, Fe3O4@CS MCNCs have been successfully prepared by an easily accessible one-pot solvothermal strategy. The as-prepared materials carry forward the virtues of being easy-to-prepare, being cost-effective, and being readily separated and dispersed. Due to the high amount of coated CS, good performance of glycopeptide enrichment with high selectivity, low detection limit and high binding capacity could be obtained by using Fe3O4@CS MCNCs. Moreover, the prepared material presented great potential in detection and identification with low abundant glycopeptides in minutes of biological samples. Additionally, it could be extended to the design of other functional magnetic based materials with colloidal nanocrystal clusters structures by one-pot method for drug delivery system, biological sensor, and so on.
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Acknowledgements
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This work were supported by China State Key Basic Research Program Grant (2013CB911202), Creative Research Group Project
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of NSFC (21321064), National Natural Sciences Foundation of China (21075105 and 21165017), and Educational Commission of the Xinjiang Uygur Autonomous Region of China (XJEDU2010I0). The funders have no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.05.037.
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References
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[1] K. Ohtsubo, J.D. Marth, Glycosylation in cellular mechanisms of health and disease, Cell 126 (2006) 855–867. [2] A. Helenius, M. Aebi, Intracellular functions of N-linked glycans, Science 291 (2001) 2364–2369. [3] R. Apweiler, H. Hermjakob, N. Sharon, On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database, Biochim. Biophys. Acta 1473 (1999) 4–8. [4] R.J. Woods, C.J. Edge, R.A. Dwek, Protein surface oligosaccharides and protein function, Nat. Struct. Mol. Biol. 1 (1994) 499–501. [5] P.T. Martin, H.H. Freeze, Glycobiology of neuromuscular disorders, Glycobiology 13 (2003) 67R–75R. [6] M.L. Montpetit, P.J. Stocker, T.A. Schwetz, J.M. Harper, S.A. Norring, L. Schaffer, S.J. North, J. Jang-Lee, T. Gilmartin, S.R. Head, Regulated and aberrant glycosylation modulate cardiac electrical signaling, Proc. Natl. Acad. Sci. 106 (2009) 16517–16522. [7] J.A. Ludwig, J.N. Weinstein, Biomarkers in cancer staging, prognosis and treatment selection, Nat. Rev. Cancer 5 (2005) 845–856. [8] D.F. Zielinska, F. Gnad, J.R. Wisniewski, M. Mann, Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints, Cell 141 (2010) 897–907. [9] I.M. Lazar, A.C. Lazar, D.F. Cortes, J.L. Kabulski, Recent advances in the MS analysis of glycoproteins: theoretical considerations, Electrophoresis 32 (2011) 3–13. [10] C.-Y. Lin, Y.-C. Ma, P.-J. Pai, G.-R. Her, A comparative study of glycoprotein concentration, glycoform profile and glycosylation site occupancy using isotope labeling and electrospray linear ion trap mass spectrometry, Anal. Chim. Acta 728 (2012) 49–56. [11] S. Feng, N. Yang, S. Pennathur, S. Goodison, D.M. Lubman, Enrichment of glycoproteins using nanoscale chelating concanavalin A monolithic capillary chromatography, Anal. Chem. 81 (2009) 3776–3783. [12] H. Zhang, X. Li, D.B. Martin, R. Aebersold, Identification and quantification of Nlinked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry, Nat. Biotechnol. 21 (2003) 660–666. [13] L. Liu, Y. Zhang, L. Zhang, G. Yan, J. Yao, P. Yang, H. Lu, Highly specific revelation of rat serum glycopeptidome by boronic acid-functionalized mesoporous silica, Anal. Chim. Acta 753 (2012) 64–72. [14] P.J. Boersema, S. Mohammed, A.J. Heck, Hydrophilic interaction liquid chromatography (HILIC) in proteomics, Anal. Bioanal. Chem. 391 (2008) 151–159. [15] L.H. Reddy, J.L. Arias, J. Nicolas, P. Couvreur, Magnetic nanoparticles: design and characterization, toxicity and biocompatibility, pharmaceutical and biomedical applications, Chem. Rev. 112 (2012) 5818–5878. [16] Z. Xiong, L. Zhao, F. Wang, J. Zhu, H. Qin, R. a. Wu, W. Zhang, H. Zou, Synthesis of branched PEG brushes hybrid hydrophilic magnetic nanoparticles for the selective enrichment of N-linked glycopeptides, Chem. Commun. 48 (2012) 8138–8140. [17] C.H. Yeh, S.H. Chen, D.T. Li, H.P. Lin, H.J. Huang, C.I. Chang, W.L. Shih, C.L. Chern, F.K. Shi, J.L. Hsu, Magnetic bead-based hydrophilic interaction liquid chromatography for glycopeptide enrichments, J. Chromatogr. A 1224 (2012) 70–78. [18] J. Gao, X. Ran, C. Shi, H. Cheng, T. Cheng, Y. Su, One-step solvothermal synthesis of highly water-soluble, negatively charged superparamagnetic Fe3O4 colloidal nanocrystal clusters, Nanoscale 5 (2013) 7026–7033. [19] D. Li, J. Tang, C. Wei, J. Guo, S. Wang, D. Chaudhary, C. Wang, Doxorubicinconjugated mesoporous magnetic colloidal nanocrystal clusters stabilized by polysaccharide as a smart anticancer drug vehicle, Small 8 (2012) 2690–2697. [20] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 31 (2006) 603–632. [21] J.-K. Francis Suh, H.W. Matthew, Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review, Biomaterials 21 (2000) 2589–2598. [22] Y. Wada, M. Tajiri, S. Yoshida, Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics, Anal. Chem. 76 (2004) 6560–6565. [23] L. Yu, X. Li, Z. Guo, X. Zhang, X. Liang, Hydrophilic interaction chromatography based enrichment of glycopeptides by using click maltose: a matrix with high selectivity and glycosylation heterogeneity coverage, Chem. Eur. J. 15 (2009) 12618–12626.
Please cite this article in press as: C. Fang, et al., One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.037
359 360 361 362 363
366
368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412
G Model
ACA 233281 1–7 C. Fang et al. / Analytica Chimica Acta xxx (2014) xxx–xxx
413 414 415 416 417 418 419 420 421 422 423 424 425 426
[24] M.H. Selman, M. Hemayatkar, A.M. Deelder, M. Wuhrer, Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides, Anal. Chem. 83 (2011) 2492–2499. [25] Z. Xiong, H. Qin, H. Wan, G. Huang, Z. Zhang, J. Dong, L. Zhang, W. Zhang, H. Zou, Layer-by-layer assembly of multilayer polysaccharide coated magnetic nanoparticles for the selective enrichment of glycopeptides, Chem. Commun. 49 (2013) 9284–9286. [26] H. Huang, Y. Jin, M. Xue, L. Yu, Q. Fu, Y. Ke, C. Chu, X. Liang, A novel click chitooligosaccharide for hydrophilic interaction liquid chromatography, Chem. Commun. 45 (2009) 6973–6975. [27] J. Ge, Y. Hu, M. Biasini, W.P. Beyermann, Y. Yin, Superparamagnetic magnetite colloidal nanocrystal clusters, Angew. Chem. Int. Ed. 46 (2007) 4342–4345. [28] B. Luo, S. Xu, A. Luo, W.-R. Wang, S.-L. Wang, J. Guo, Y. Lin, D.-Y. Zhao, C.-C. Wang, Mesoporous biocompatible and acid-degradable magnetic colloidal nanocrystal clusters with sustainable stability and high hydrophobic drug loading capacity, ACS Nano 5 (2011) 1428–1435. [29] C. Cheng, Y. Wen, X. Xu, H. Gu, Tunable synthesis of carboxyl-functionalized magnetite nanocrystal clusters with uniform size, J. Mater. Chem. 19 (2009) 8782–8788.
7
[30] C. Kuo, I. Wu, H. Hsiao, K. Khoo, Rapid glycopeptide enrichment and Nglycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles, Anal. Bioanal. Chem. 402 (2012) 2765–2776. [31] M. Thaysen-Andersen, I.B. Thøgersen, H.J. Nielsen, U. Lademann, N. Brünner, J.J. Enghild, P. Højrup, Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1, Mol. Cell. Proteomics 6 (2007) 638–647. [32] P. Hägglund, J. Bunkenborg, F. Elortza, O.N. Jensen, P. Roepstorff, A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation, J. Proteome. Res. 3 (2004) 556–566. [33] G. Huang, Z. Xiong, H. Qin, J. Zhu, Z. Sun, Y. Zhang, X. Peng, H. Zou, Synthesis of zwitterionic polymer brushes hybrid silica nanoparticles via controlled polymerization for highly efficient enrichment of glycopeptides, Anal. Chim. Acta 809 (2014) 61–68. [34] J. Zhu, F. Wang, R. Chen, K. Cheng, B. Xu, Z. Guo, X. Liang, M. Ye, H. Zou, Centrifugation assisted microreactor enables facile integration of trypsin digestion, hydrophilic interaction chromatography enrichment, and oncolumn deglycosylation for rapid and sensitive N-glycoproteome analysis, Anal. Chem. 84 (2012) 5146–5153.
Please cite this article in press as: C. Fang, et al., One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.037
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