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Thiol-ene click synthesis of L-Cysteine-bonded zwitterionic hydrophilic magnetic nanoparticles for selective and efficient enrichment of glycopeptides
Talanta 160 (2016) 461–469
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Thiol-ene click synthesis of L-Cysteine-bonded zwitterionic hydrophilic magnetic nanoparticles for selective and efficient enrichment of glycopeptides Runqing Wu a, Yang Xie b,n, Chunhui Deng a,n a Department of Chemistry and Institutes of Biomedical Sciences, Collaborative Innovation Center of Genetics and Development, Fudan University, Shanghai 200433, P.R. China b Department of Orthopaedic Surgery, Second Military Medical University, The Affiliated Changhai Hospital of Shanghai 200433, P.R. China
art ic l e i nf o
a b s t r a c t
Article history: Received 27 April 2016 Received in revised form 16 July 2016 Accepted 22 July 2016 Available online 25 July 2016
Efficient and specific enrichment of low-abundant glycopeptides from complex biological samples is essential to glycoproteomics analysis. Herein, novel magnetic zwitterionic hydrophilic nanoparticles based on thiol-ene click chemistry were synthesized. The functionalized magnetic nanoparticles exhibited superior performance in glycopeptide enrichment of HRP tryptic digest, demonstrating low detection limit (0.04 ng/μL), high selectivity (a mixture of HRP and BSA at the mass ratio of 1:50) and reproducibility (5 repeating cycles). In addition, the material was successfully applied to glycopeptide enrichment from human serum. The outstanding results indicate the potential of the method in the development of glycoproteomics analysis in real biological samples. & 2016 Elsevier B.V. All rights reserved.
Keywords: Thiol-ene click chemistry MALDI-TOF-MS Glycopeptide Hydrophilicity Enrichment
1. Introduction Glycoproteomics analysis has attracted increasing attention in recent years due to the crucial role in biology and diseases. As one of the most important and complicated post-translational modifications (PTMs), protein glycosylation is associated with various biological processes, including cell signal transduction [1], intracellular sorting [2], and molecular recognition [3] and regulation of protein folding [4]. In addition, glycoproteins have been successfully employed as clinical biomarkers on account of the fast and dynamic changes in protein glycosylation [5,6]. Highly selective identification of glycoproteins is of great significance. Recently, mass spectrometry (MS)-based technique has been widely used in glycoproteomics analysis with the advantages of sequence recognition, quick detection, high accuracy and simple operation [7–12]. Nevertheless, the analysis of protein glycosylation still remains many challenges due to the low abundance, severe signal interference from non-glycopeptide, poor ionization efficiency and glycan structure intricacy. Therefore, separation and enrichment of glycopeptides from complex samples are necessary before MS analysis. Currently, some strategies have been applied to glycopeptide n
Corresponding authors. E-mail addresses: [email protected] (Y. Xie), [email protected] (C. Deng).
enrichment from complex biological samples, including hydrazide chemistry [13–17], lectin affinity chromatography [18–20], boronate affinity chromatography [21–23], titanium dioxide [24] and hydrophilic-interaction liquid chromatography (HILIC) [25–28]. Compared with other enriching strategies, hydrophilic-interaction liquid chromatography (HILIC) method is widely used in separation and identification of glycopeptides. HILIC method has unique advantages, such as rapid and efficient enrichment procedure, reservation of glycan structural information, good reproducibility, simple reaction condition and great compatibility with mass spectrometry [29]. Due to the associated glycan moieties, glycopeptides exhibit better hydrophilicity than non-glycopeptides. Several functional HILIC-based materials have been reported for glycopeptide enrichment, containing maltose, glucose, cotton, polyethylene glycol, chitosan, sepharose, cyclodextrins and metal organic frameworks [26,30–37]. In addition to conventional HILIC, zwitterionic-HILIC (ZIC-HILIC) materials show better performance in glycopeptide enrichment due to the excellent hydrophilicity enhanced by co-existed negative and positive functional groups [31,38,39]. Therefore, it is necessary to develop ZIC-HILIC materials for highly selective and efficient enrichment of glycopeptides. In this work, a novel ZIC-HILIC nanomaterial (Fe3O4@SiO2@LCys nanoparticles) was prepared for glycopeptide enrichment in complicated biological samples via thiol-ene click chemistry. Magnetic Fe3O4 nanoparticles were synthesized using solvothermal reaction for quick magnetic response and good
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biocompatibility [40]. After that, a core-shell structure was accomplished by coating a SiO2 layer outside through a sol-gel approach to obtain good hydrophilicity, stabilize the surface and prevent the aggregation of Fe3O4 nanoparticles [41]. A silane coupling reagent was modified on the surface subsequently in order to provide large numbers of carbon-carbon double bonds. The final products of Fe3O4@SiO2@L-Cys nanoparticles were synthesized based on the thiol-ene click reaction between the thiol groups in L-Cysteine and carbon-carbon double bonds [42,43]. As a ZIC-HILIC stationary phase, L-Cysteine possesses both negative and positive functional groups (–COOH and –NH2), greatly improving the hydrophilicity of the material [44–46]. The resultant hydrophilic magnetic nanoparticles were successfully applied to highly selective and efficient glycopeptide enrichment from standard glycoprotein tryptic digest and human serum, indicating the Fe3O4@SiO2@L-Cys nanoparticles are promising materials for glycopeptide analysis in complex biological samples.
2. Material and methods 2.1. Chemicals and materials Iron (Ⅲ) chloride hexahydrate (FeCl3 6H2O), tetraethyl orthosilicate (TEOS), ammonium hydroxide (NH3 H2O, 28 wt%), ethylene glycol, sodium acetate and ammonium bicarbonate (NH4HCO3) were purchased from Shanghai Chemical Corp. Vinyltriethoxysilane (VTES), L-Cysteine, horseradish peroxidase (HRP), bovine serum albumin (BSA) were supplied by Sigma-Aldrich. PNGase F was purchased from New England Biolabs (Ipswich, MA, USA). Human serum was provided by Zhongshan Hospital (Shanghai). Ultrapure water (18.2 MΩ cm) was used for all experiments, purified by a Milli-Q system (Millipore, Bedford, MA). Acetonitrile (ACN) was obtained from Merck (Darmstadt, Germany). All other reagents were of analytical grade. 2.2. Synthesis of Fe3O4 nanoparticles Fe3O4 nanoparticles were synthesized through a hydrothermal method. Specifically, 1.35 g FeCl3 6H2O was dissolved into 75 mL ethylene glycol under magnetic stirring. 3.6 g sodium acetate and 0.2 g anhydrous sodium citrate were added into the mixture, followed by vigorously stirring for 0.5 h. The obtained yellow solution was then transferred to a Teflon-lined stainless steel autoclave. After reacting at 200 °C for 16 h and cooling to room temperature, the obtained Fe3O4 nanoparticles were separated from the solution by a magnet. Then the Fe3O4 nanoparticles were washed with ethanol and deionized water and dried in vacuum at 50 °C overnight. 2.3. Synthesis of Fe3O4@SiO2 nanoparticles Obtained Fe3O4 nanoparticles (0.18 g) were dispersed in a mixed solution containing 160 mL ethanol, 1 mL deionized water and 2 mL NH3 H2O under mechanical stirring for 0.5 h at 30 °C. Subsequently, 1.0 mL tetraethyl orthosilicate (TEOS) was added dropwise within 3 min. The reaction was stirred vigorously at 30 °C for 10 h. After washing with ethanol and deionized water for three times, the resulted Fe3O4@SiO2 nanoparticles were vacuumdried at 50 °C for further use. 2.4. Synthesis of Fe3O4@SiO2@L-Cys nanoparticles The prepared 50 mg Fe3O4@SiO2 nanoparticles were dispersed in 100 mL isopropyl alcohol solution. Then, 2 mL vinyltriethoxysilane (VTES) was added in the mixture under nitrogen
atmosphere for 0.5 h. The mixed solution was mechanical stirred at room temperature for 16 h. The VTES-modified Fe3O4@SiO2 microspheres were collected and washed with ethanol and deionized water repeatedly, and redispersed in ethanol (90 mL) in a dried single-necked flask. The mixture of 50 mg L-Cysteine in water (20 mL) and 2 mg azoisobutyronitrile (AIBN) in ethanol (2 mL) was added in the flask under ultrasonic condition. The resulting mixture was then heated at 60 °C with mechanical stirring for 3 h. The obtained Fe3O4@SiO2@L-Cys microspheres were washed with ethanol and deionized water for three times by magnetic separation and dried in vacuum at 50 °C. 2.5. Characterization Fourier-transform infrared (FT-IR) spectra was obtained on Nicolet Fourier spectrophotometer using KBr pellets (Thermo Fisher, USA). Transmission electron microscopy (TEM) images were collected on JEOL2011 microscope (Japan). Scanning electron microscopy (SEM) images were performed on a Philips XL30 electron microscope (Netherlands). The saturation magnetization curve was monitored by MPMS (SQUID) VSM (Quantum Design, San Diego, USA) at room temperature. Powder X-ray diffraction (XRD) patterns were performed on Bruker D4 X-ray diffractometer with Ni-filtered Cu Kα radiation (40 kV, 40 mA). 2.6. Tryptic digestion of the glycoproteins and human serum The tryptic digestion is a mature and universal methodology approach and has been reported in previous studies [26–28]. 2 mg HRP was dissolved in 1 mL buffer containing 25 mM ammonium bicarbonate (pH 7.9) and denatured in boiling water for 5 min. After reducing in 10 mM DTT at 60 °C for 0.5 h, the protein was alkylated in the dark at 37 °C for 1 h by adding 20 mM IAA. Then the solution was digested with trypsin at an enzyme/protein ratio of 1:40 (w/w) at 37 °C for 16 h. The digest was stored at 20 °C for further use. For tryptic digestion of human serum, the sample was first centrifuged at 12,000 rpm for 10 min. The obtained supernatant (2 μL) was a hundredfold diluted with 25 mM ammonium bicarbonate (pH 7.9) and denatured at 100 °C for 5 min. The mixture was reduced in DTT (10 mM) and then alkylated with IAA (25 mM) in the dark at 37 °C for 1 h. Subsequently, the mixture was digested by trypsin at an enzyme/protein ratio of 1:40 (w/w) at 37 °C for 16 h. The tryptic digestion was lyophilized and stored at 20 °C for further use. 2.7. Enrichment of glycopeptides Fe3O4@SiO2@L-Cys microspheres (150 μg) were washed with loading buffer (90% ACN/H2O, 0.1% TFA) and diluted with 100 μL loading buffer containing 0.4 μg HRP tryptic digestion. The mixture were shaked mildly at 37 °C for 30 min, and then wash with 100 μL loading buffer to remove the non-glycopeptides for three times with the aid of an external magnetic field. The captured glycopeptides were incubated in 10 μL eluting buffer (30% ACN/ H2O, 0.1% TFA) under gentle agitation for 30 min at 37 °C. The elute was detected by MALDI-TOF-MS. For the glycopeptide enrichment from human serum, the lyophilized tryptic digest was dissolved in 200 μL loading buffer. 1 mg Fe3O4@SiO2@L-Cys microspheres were added in the solution and incubated under mild vibration for 30 min at 37 °C. After washing with loading buffer for three times, the mixture were dispersed in eluting buffer under mild vibration for another 30 min at 37 °C. The obtained glycopeptides were lyophilized and redissolved in 25 mM ammonium bicarbonate (pH 7.9). Then, 500 U of PNGase F was added and the mixture was incubated for 12 h at 37 °C to
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remove the glycan moieties. The deglycosylated peptides were evaporated to dryness for further LC-MS analysis. 2.8. Mass spectrometry analysis All the MALDI-TOF-MS experiments were operated in a reflector positive mode on a 5800 Proteomics Analyzer (Applied Biosystems, USA). The Nd/YAG laser was 355 nm, the frequency was 200 Hz and acceleration voltage was 20 kV. The 2, 5-dihydroxybenzoic acid (DHB, 20 mg) was dissolved in 1 mL mixed solution of ACN/H2O/TFA (50:50:0.1, v/v/v) to prepare a matrix. 1 μL sample solution was deposited onto the MALDI plate, followed by dropping 1 μL DHB matrix solution for MS analysis.
3. Results and discussion 3.1. Synthesis and characterization of Fe3O4@SiO2@L-Cys nanoparticles The protocol for the preparation of Fe3O4@SiO2@L-Cys microspheres was illustrated in Scheme 1. Fe3O4 nanoparticles were firstly synthesized via a modified hydrothermal reaction based on a high temperature reduction of Iron(Ⅲ) chloride hexahydrate in the presence of trisodium citrate. Then, a SiO2 layer was formed on the surface of Fe3O4 nanoparticles via sol-gel approach.
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Subsequently, Vinyltriethoxysilane (VTES) was used for the surface modification of Fe3O4@SiO2 nanoparticles to endow abundant carbon-carbon double bonds. Fe3O4@SiO2@L-Cys microspheres were achieved by the thiol-ene click reaction. The thiol-ene click reaction can preserve amino and carboxyl groups in the L-Cysteine, maintaining the superior zwitterionic hydrophilicity. The size and morphology of Fe3O4@SiO2@L-Cys nanoparticles were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). As shown in Fig. 1, the Fe3O4 nanoparticles were uniform in both size and shape, with a mean diameter of about 200 nm. After being coated with SiO2, the size of core-shell Fe3O4@SiO2 nanoparticles was increased to about 240 nm (Fig. 1a). The modification with L-Cysteine on the surface of Fe3O4@SiO2 resulted in an obvious agglomeration, indicating the successful coating of L-Cysteine layer (Fig. 1b). The SEM images of Fe3O4@SiO2 and Fe3O4@SiO2@L-Cys showed spherical shape with little difference in size and morphology (Fig. 1c and d). Energy Dispersive Spectrometer (EDS) analysis of the Fe3O4@SiO2@L-Cys nanoparticles was shown in Table S1. The S element in the L-Cysteine was confirmed, indicating that L-Cysteine was successfully grafted on the surface of silica shell. Fourier transform infrared (FT-IR) spectroscopy was used to confirm the chemical composition of Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2@L-Cys nanoparticles. As shown in Fig. 2a, the peak at 567 cm 1 was assigned to the Fe–O stretching vibration. In the FTIR spectrum of Fe3O4@SiO2 nanoparticles (Fig. 2b), absorption
Scheme 1. Schematic illustrations of the (a) synthetic procedure of the Fe3O4@SiO2@L-Cys nanoparticles and (b) selective glycopeptide enrichment strategy by Fe3O4@SiO2@L-Cys nanoparticles.
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Fig. 1. (a) TEM and (c) SEM images of Fe3O4@SiO2; (b) TEM and (d) SEM images of Fe3O4@SiO2@L-Cys nanoparticles.
Fig. 2. FT-IR spectra of (a) Fe3O4, (b) Fe3O4@SiO2 and (c) Fe3O4@SiO2@L-Cys nanoparticles.
Fig. 3. Magnetic hysteresis curves (c) Fe3O4@SiO2@L-Cys nanoparticles.
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of
(a)
Fe3O4,
(b)
Fe3O4@SiO2
and
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Fig. 4. MALDI-TOF-MS spectra of 4 ng/μL HRP tryptic digest (a) direct analysis; after enrichment by (b) Fe3O4@SiO2@L-Cys, (c) Fe3O4 and (d) Fe3O4@SiO2 nanoparticles. Glycopeptides were marked with numbers in the spectra and detailed information was shown in Table 1.
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Table 1 The detailed information of glycopeptides derived from tryptic digest of HRP after enrichment by Fe3O4@SiO2@L-Cys nanoparticles. Number
m/z
Glycan composition
Peptide sequence
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
2069 2541 2591 2612 2850 3088 3322 3353 3524 3607 3671 3895 4058 4224 4836
PNVSN#IVR SSPN#ATDTIPLVR PTLN#TTYLQTLR MGN#ITPLTGTQGQIR GLIQSDQELFSSPN#ATDTIPLVR GLCPLNGN#LSALVDFDLR QLTPTFYDNSCPN#VSNIVR SFAN#STQTFFNAFVEAMDR GLIQSDQELFSSPN#ATDTIPLVR NQCRGLCPLNGN#LSALVDFDLR GLIQSDQELFSSPN#ATDTIPLVR LHFHDCFVNGCDASILLDN#TTSFR QLTPTFYDNSC(AAVESACPR)PN#VSNIVR-H2O QLTPTFYDNSC(AAVESACPR)PN#VSNIVR LYN#FSNTGLPDPTLN#TTYLQTLR
16
4985
[Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Xyl]1 [HexNAc]1[Fuc]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1
LYN#FSNTGLPDPTLN#TTYLQTLR
HexNAc¼N-acetylglucosamine, Hex ¼mannose, Fuc¼ fructose, Xyl¼ xylose. N# denotes the N-linked glycosylation site.
peaks at 1088 and 954 cm 1 were separately attributed to the Si– O–Si and Si–OH stretching vibration. In the FT-IR spectrum of Fe3O4@SiO2@L-Cys nanoparticles (Fig. 2c), new absorption peak at 1640 cm 1 was ascribed to the stretching vibration of C ¼O bond, and the broad peak at around 3300 cm 1 was assigned to the N–H stretching vibration. The X-ray diffraction patterns (XRD) of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@L-Cys nanoparticles were shown in Fig. S1. The characteristic diffraction peaks of Fe3O4 were 30.0°, 35.5°, 42.9°, 57.1° and 62.6°, corresponding to the 2θ values of (220), (311), (400), (511) and (440), respectively. The XRD results were in good agreement with the face-centered cubic phase of Fe3O4 (JCPDS Card no. 19-629), demonstrating that the structure of the nanoparticles remains unchanged after modification. The magnetic properties of the synthesized Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@L-Cys nanoparticles were investigated by a vibrating sample magnetometer (VSM) at room temperature. As shown in Fig. 3, the saturation magnetic (MS) values of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@L-Cys nanoparticles were 81.74, 64.89 and 60.01 emu/g, respectively. Due to superior superparamagnetic properties, Fe3O4@SiO2@L-Cys nanoparticles could be easily separated from solution within several seconds with the help of a magnet. The nanoparticles redispersed quickly in the solution after removing the magnet, which could be applied to the efficient enrichment and separation of glycopeptides. In addition, the hydrophilicity of the core-shell magnetic nanoparticles was evaluated by analyzing the water contact angle using the powder tableting method. As shown in Fig. S2, the water contact angles of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@L-Cys nanoparticles were 12.8°, 22.8° and 22.7°, respectively. The relatively small water contact angles demonstrated the excellent hydrophilicity of as-prepared nanoparticles, indicating promising hydrophilic enrichment of glycopeptides. 3.2. Selective enrichment of glycopeptides by Fe3O4@SiO2@L-Cys nanoparticles As a typical glycoprotein with 9 glycosylation sites, horseradish peroxidase (HRP), has been extensively studied and applied to glycoproteomics analysis [21–23,26,27]. Therefore, HRP tryptic digest was employed as a standard biological sample to evaluate the glycopeptide enrichment efficiency of the synthesized Fe3O4@SiO2@L-Cys nanoparticles. The enrichment strategy
included three steps: incubation for enriching glycopeptides, wash for removing non-glycopeptides and elution for releasing captured glycopeptides. Then, the obtained glycopeptides were detected by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). As shown in Fig. 4a, only one glycopeptide could be observed in direct analysis of 4 ng/μL HRP tryptic digest owing to the severe signal suppression from co-existed non-glycopeptides. However, after enrichment by Fe3O4@SiO2@L-Cys nanoparticles, 16 peaks corresponding to the glycopeptides clearly dominated the spectrum (Fig. 4b), with greatly improved intensity and signal-to-noise (S/N) ratios. Detailed information about the captured glycopeptides was shown in Table 1. Moreover, signals of non-glycopeptides almost disappeared. To verify the hydrophilic enrichment capacity of the material, the efficiency of glycopeptide enrichment were compared by equivalent amount of Fe3O4 and Fe3O4@SiO2 nanoparticles. As the mass spectrum revealed, only 3 glycopeptides was detected with the enrichment of Fe3O4 nanoparticles and 4 peaks of glycopeptides were obtained with the enrichment of Fe3O4@SiO2 nanoparticles (Fig. 4c and d). With excellent hydrophilicity, Fe3O4@SiO2@L-Cys nanoparticles showed better performance in glycopeptide enrichment. The result was attributed to the modification of highly abundant L-Cysteine, which could significantly enhance hydrophilicity of the material with zwitterionic functional groups (–NH2 and –COOH). To evaluate the sensitivity of the glycopeptide enrichment, different concerntration of HRP tryptic digests (2 ng/μL and 0.04 ng/μL) were tested (Fig. 5a and b). As shown in Fig. 5b, when the concerntration of HRP tryptic digest was as low as 0.04 ng/μL, three peaks assigned to glycopeptides were still detected with clean background and high intensity. The selectivity of this method was investigated by the mixture of HRP (standard glycoprotein) and BSA (standard non-glycoprotein) tryptic digest at a mass ratio of 1:50 (HRP: BSA). Due to the serious interference by the non-glycopeptides of BSA tryptic digest, the signals of glycopeptides were hardly detected directly (Fig. 5c). However, as shown in Fig. 5d, 9 glycopeptide peaks with significantly improved intensity appeared after enrichment by Fe3O4@SiO2@L-Cys nanoparticles. The results indicated that the zwitterionic hydrophilic method can enrich glycopeptides with remarkable sensitivity and selectivity. The reproducibility of the Fe3O4@SiO2@L-Cys nanoparticles was also examined using the method. The enriching procedure of
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Fig. 5. MALDI-TOF-MS spectra of (a) 2 ng/μL and (b) 0.04 ng/μL HRP tryptic digest after enrichment by Fe3O4@SiO2@L-Cys nanoparticles. MALDI-TOF-MS spectra of the tryptic digest mixture of HRP and BSA (with a mass ratio of 1:50): (c) direct analysis; (d) after enrichment by Fe3O4@SiO2@L-Cys nanoparticles. Glycopeptides were marked with numbers in the spectra and detailed information was shown in Table 1.
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Fig. 6. MALDI-TOF-MS spectra of 4 ng/μL HRP tryptic digest after enrichment by Fe3O4@SiO2@L-Cys nanoparticles for (a) the third time and (b) the fifth time. Glycopeptides were marked with numbers in the spectra and detailed information was shown in Table 1.
glycopeptides was repeated for five times with the same Fe3O4@SiO2@L-Cys nanoparticles. Before each enriching cycle, the Fe3O4@SiO2@L-Cys nanoparticles were washed sequentially with eluting and loading buffers for several times to remove the adsorbed peptides on the surface of nanoparticles and other impurities. After third and fifth enriching cycle, the obtained signals of glycopeptides were as similar as the first time (Fig. 6), demonstrating the stability and enrichment efficiency of the Fe3O4@SiO2@L-Cys nanoparticles. The practical application of the method was further investigated by detecting glycopeptides and proteins of the human serum sample. The enriching procedure was the same as standard glycopeptide enrichment. The eluted glycopeptides were incubated with PNGase F for deglycosylation. Then the glycosylated peptides were analyzed by LC-MS/MS. A total of 199 unique N-glycosylation sites assigned to 88 glycoproteins were identified (Table S2), indicating the successful application to real biological samples by the method.
4. Conclusion In summary, a novel and efficient method for glycopeptide enrichment was established by fabricating a zwitterionic hydrophilic magnetic core-shell material (Fe3O4@SiO2@L-Cys nanoparticles). With abundant zwitterionic functional groups, L-Cysteine was immobilized based on the thiol-ene click chemistry, distinctly enhancing the hydrophilicity of the material. The Fe3O4@SiO2@L-Cys nanoparticles exhibited excellent performance in HILIC enrichment of glycopeptide, such as high sensitivity, selectivity and reproducibility. Furthermore, the Fe3O4@SiO2@L-Cys nanoparticles were successfully applied to glycopeptide enrichment from human serum sample. In view of these advantages, the innovative method shows a great potential for glycoproteomics analysis in complex biological samples.
Notes The authors declare no competing financial interest.
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Acknowledgement Financial support is gratefully acknowledged from the Research Fund for the Doctoral Program of Higher Education of China (20110071110007, 20100071120053), the National Natural Science Foundation of China (21075022, 20875017, 21105016), the National Basic Research Priorities Program of China (2012CB910602, 2013CB911201), the Shanghai Municipal Natural Science Foundation (11ZR1403200), and Shanghai Leading Academic Discipline Project (B109).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2016.07. 045.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
G. Durand, N. Seta, Clin. Chem. 46 (2000) 795–805. K. Ohtsubo, J.D. Marth, Cell 126 (2006) 855–867. G.W. Hart, R.J. Copeland, Cell 143 (2010) 672–676. M.A. Daniels, K.A. Hogquist, S.C. Jameson, Nat. Immunol. 3 (2002) 903–910. Y. Tian, H. Zhang, Proteom.: Clin. Appl. 4 (2010) 124–132. S.A. Svarovsky, L. Joshi, Anal. Methods 6 (2014) 3918–3936. C. Lei, K. Qian, O. Noonan, A. Nouwens, C. Yu, Nanoscale 5 (2013) 12033–12042. A. Dell, H.R. Morris, Science 291 (2001) 2351–2356. D. Baycin-Hizal, Y. Tian, I. Akan, E. Jacobson, D. Clark, A. Wu, R. Jampol, K. Palter, M. Betenbaugh, H. Zhang, Anal. Chem. 83 (2011) 5296–5303. S. Pan, R. Chen, R. Aebersold, T.A. Brentnall, Mol. Cell. Proteom. 10 (2011) 1–14. Y. Yu, J. Fournier, M. Gilar, J.C. Gebler, Anal. Chem. 79 (2007) 1731–1738. Y. Zhang, B.R. Fonslow, B. Shan, M.C. Baek, J.R. Yates III, Chem. Rev. 113 (2013) 2343–2394. Z. Zhang, D. Sun, Y. Cong, J. Mao, J. Huang, H. Qin, J. Liu, G. Huang, L. Wang, M. Ye, H. Zou, J. Proteom. Res. 14 (2015) 3892–3899. L. Liu, M. Yu, Y. Zhang, C. Wang, H. Lu, ACS Appl. Mater. Interfaces 6 (2014) 7823–7832. Z. Zhang, Z. Sun, J. Zhu, J. Liu, G. Huang, M. Ye, H. Zou, Anal. Chem. 86 (2014) 9830–9837. J. Huang, H. Wan, Y. Yao, J. Li, K. Cheng, J. Mao, J. Chen, Y. Wang, H. Qin, W. Zhang, M. Ye, H. Zou, Anal. Chem. 87 (2015) 10199–10204. Y. Zhang, M. Yu, C. Zhang, Y. Wang, Y. Di, C. Wang, H. Lu, Chem. Commun. 51 (2015) 5982–5985. J.Y. Kim, D. Oh, S.K. Kim, D. Kang, M.H. Moon, Anal. Chem. 86 (2014) 7650–7657. J.A. Ferreira, A.L. Daniel-da-Silva, R.M. Alves, D. Duarte, I. Vieira, L.L. Santos,
469
R. Vitorino, F. Amado, Anal. Chem. 83 (2011) 7035–7043. [20] Z. Tan, H. Yin, S. Nie, Z. Lin, J. Zhu, M.T. Ruffin, M.A. Anderson, D.M. Simeone, D. M. Lubman, J. Proteom. Res. 14 (2015) 1968–1978. [21] X. Zhang, J. Wang, X. He, L. Chen, Y. Zhang, ACS Appl. Mater. Interfaces 7 (2015) 24576–24584. [22] Y. Wang, M. Liu, L. Xie, C. Fang, H. Xiong, H. Lu, Anal. Chem. 86 (2014) 2057–2064. [23] Y. Zhang, W. Ma, D. Li, M. Yu, J. Guo, C. Wang, Small 10 (2014) 1379–1386. [24] M.R. Larsen, S.S. Jensen, L.A. Jakobsen, N.H. Heegaard, Mol. Cell. Proteom. 6 (2007) 1778–1787. [25] T. Jin, Z. Xiong, X. Zhu, N. Mehio, Y. Chen, J. Hu, W. Zhang, H. Zou, H. Liu, S. Dai, ACS Macro Lett. 4 (2015) 570–574. [26] C. Bi, Y. Zhao, L. Shen, K. Zhang, X. He, L. Chen, Y. Zhang, ACS Appl. Mater. Interfaces 7 (2015) 24670–24678. [27] B. Jiang, Q. Wu, N. Deng, Y. Chen, L. Zhang, Z. Liang, Y. Zhang, Nanoscale 8 (2016) 4894–4897. [28] J. Li, F. Wang, J. Liu, Z. Xiong, G. Huang, H. Wan, Z. Liu, K. Cheng, H. Zou, Chem. Commun. 51 (2015) 4093–4096. [29] H. Lin, J. Ou, Z. Zhang, J. Dong, M. Wu, H. Zou, Anal. Chem. 84 (2012) 2721–2728. [30] L. Yu, X. Li, Z. Guo, X. Zhang, X. Liang, Chem. Eur. J. 15 (2009) 12618–12626. [31] X. Zou, J. Jie, B. Yang, Chem. Commun. 52 (2016) 3251–3253. [32] J. Zheng, Y. Xiao, L. Wang, Z. Lin, H. Yang, L. Zhang, G. Chen, J. Chromatogr. A 1358 (2014) 29–38. [33] Y. Zhao, L. Yu, Z. Guo, X. Li, X. Liang, Anal. Bioanal. Chem. 399 (2011) 3359–3365. [34] Y.W. Zhang, Z. Li, Q. Zhao, Y.L. Zhou, H.W. Liu, X.X. Zhang, Chem. Commun. 50 (2014) 11504–11506. [35] M.H.J. Selman, M. Hemayatkar, A.M. Deelder, M. Wuhrer, Anal. Chem. 83 (2011) 2492–2499. [36] P. Dedvisitsakul, S. Jacobsen, B. Svensson, J. Bunkenborg, C. Finnie, P. Hagglund, J. Proteom. Res. 13 (2014) 2696–2703. [37] Z. Xiong, L. Zhao, F. Wang, J. Zhu, H. Qin, R. Wu, W. Zhang, H. Zou, Chem. Commun. 48 (2012) 8138–8140. [38] Y. Chen, Z. Xiong, L. Zhang, J. Zhao, Q. Zhang, L. Peng, W. Zhang, M. Ye, H. Zou, Nanoscale 7 (2015) 3100–3108. [39] M. Pabst, I. Benesova, S.R. Fagerer, M. Jacobsen, K. Eyer, G. Schmidt, R. Steinhoff, J. Krismer, F. Wahl, J. Preisler, R. Zenobi, J. Proteom. Res. 15 (2016) 326–331. [40] Y. Deng, Y. Cai, Z. Sun, J. Liu, C. Liu, J. Wei, W. Li, C. Liu, Y. Wang, D. Zhao, J. Am. Chem. Soc. 132 (2010) 8466–8473. [41] X. Xu, C. Deng, M. Gao, W. Yu, P. Yang, X. Zhang, Adv. Mater. 18 (2006) 3289–3293. [42] D. Yu, A. Shen, Z. Guo, Y. Yan, J. Yan, G. Jin, X. Liang, Chem. Commun. 51 (2015) 14778–14780. [43] I. Tijunelyte, E. Guenin, N. Lidgi-Guigui, F. Colas, J. Ibrahim, T. Toury, M. Lamy de la Chapelle, Nanoscale 8 (2016) 7105–7112. [44] B. Jiang, Y. Liang, Q. Wu, H. Jiang, K. Yang, L. Zhang, Z. Liang, X. Peng, Y. Zhang, Nanoscale 6 (2014) 5616–5619. [45] E. Ataman, C. Isvoranu, J.N. Andersen, J. Schnadt, K. Schulte, J. Phys. Chem. Lett. 2 (2011) 1677–1681. [46] Y. Liang, C. Wu, Q. Zhao, Q. Wu, B. Jiang, Y. Weng, Z. Liang, L. Zhang, Y. Zhang, Anal. Chim. Acta 900 (2015) 83–89.
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Thiol-ene click synthesis of L-Cysteine-bonded zwitterionic hydrophilic magnetic nanoparticles for selective and efficient enrichment of glycopeptides Runqing Wu a, Yang Xie b,n, Chunhui Deng a,n a Department of Chemistry and Institutes of Biomedical Sciences, Collaborative Innovation Center of Genetics and Development, Fudan University, Shanghai 200433, P.R. China b Department of Orthopaedic Surgery, Second Military Medical University, The Affiliated Changhai Hospital of Shanghai 200433, P.R. China
art ic l e i nf o
a b s t r a c t
Article history: Received 27 April 2016 Received in revised form 16 July 2016 Accepted 22 July 2016 Available online 25 July 2016
Efficient and specific enrichment of low-abundant glycopeptides from complex biological samples is essential to glycoproteomics analysis. Herein, novel magnetic zwitterionic hydrophilic nanoparticles based on thiol-ene click chemistry were synthesized. The functionalized magnetic nanoparticles exhibited superior performance in glycopeptide enrichment of HRP tryptic digest, demonstrating low detection limit (0.04 ng/μL), high selectivity (a mixture of HRP and BSA at the mass ratio of 1:50) and reproducibility (5 repeating cycles). In addition, the material was successfully applied to glycopeptide enrichment from human serum. The outstanding results indicate the potential of the method in the development of glycoproteomics analysis in real biological samples. & 2016 Elsevier B.V. All rights reserved.
Keywords: Thiol-ene click chemistry MALDI-TOF-MS Glycopeptide Hydrophilicity Enrichment
1. Introduction Glycoproteomics analysis has attracted increasing attention in recent years due to the crucial role in biology and diseases. As one of the most important and complicated post-translational modifications (PTMs), protein glycosylation is associated with various biological processes, including cell signal transduction [1], intracellular sorting [2], and molecular recognition [3] and regulation of protein folding [4]. In addition, glycoproteins have been successfully employed as clinical biomarkers on account of the fast and dynamic changes in protein glycosylation [5,6]. Highly selective identification of glycoproteins is of great significance. Recently, mass spectrometry (MS)-based technique has been widely used in glycoproteomics analysis with the advantages of sequence recognition, quick detection, high accuracy and simple operation [7–12]. Nevertheless, the analysis of protein glycosylation still remains many challenges due to the low abundance, severe signal interference from non-glycopeptide, poor ionization efficiency and glycan structure intricacy. Therefore, separation and enrichment of glycopeptides from complex samples are necessary before MS analysis. Currently, some strategies have been applied to glycopeptide n
Corresponding authors. E-mail addresses: [email protected] (Y. Xie), [email protected] (C. Deng).
enrichment from complex biological samples, including hydrazide chemistry [13–17], lectin affinity chromatography [18–20], boronate affinity chromatography [21–23], titanium dioxide [24] and hydrophilic-interaction liquid chromatography (HILIC) [25–28]. Compared with other enriching strategies, hydrophilic-interaction liquid chromatography (HILIC) method is widely used in separation and identification of glycopeptides. HILIC method has unique advantages, such as rapid and efficient enrichment procedure, reservation of glycan structural information, good reproducibility, simple reaction condition and great compatibility with mass spectrometry [29]. Due to the associated glycan moieties, glycopeptides exhibit better hydrophilicity than non-glycopeptides. Several functional HILIC-based materials have been reported for glycopeptide enrichment, containing maltose, glucose, cotton, polyethylene glycol, chitosan, sepharose, cyclodextrins and metal organic frameworks [26,30–37]. In addition to conventional HILIC, zwitterionic-HILIC (ZIC-HILIC) materials show better performance in glycopeptide enrichment due to the excellent hydrophilicity enhanced by co-existed negative and positive functional groups [31,38,39]. Therefore, it is necessary to develop ZIC-HILIC materials for highly selective and efficient enrichment of glycopeptides. In this work, a novel ZIC-HILIC nanomaterial (Fe3O4@SiO2@LCys nanoparticles) was prepared for glycopeptide enrichment in complicated biological samples via thiol-ene click chemistry. Magnetic Fe3O4 nanoparticles were synthesized using solvothermal reaction for quick magnetic response and good
http://dx.doi.org/10.1016/j.talanta.2016.07.045 0039-9140/& 2016 Elsevier B.V. All rights reserved.
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biocompatibility [40]. After that, a core-shell structure was accomplished by coating a SiO2 layer outside through a sol-gel approach to obtain good hydrophilicity, stabilize the surface and prevent the aggregation of Fe3O4 nanoparticles [41]. A silane coupling reagent was modified on the surface subsequently in order to provide large numbers of carbon-carbon double bonds. The final products of Fe3O4@SiO2@L-Cys nanoparticles were synthesized based on the thiol-ene click reaction between the thiol groups in L-Cysteine and carbon-carbon double bonds [42,43]. As a ZIC-HILIC stationary phase, L-Cysteine possesses both negative and positive functional groups (–COOH and –NH2), greatly improving the hydrophilicity of the material [44–46]. The resultant hydrophilic magnetic nanoparticles were successfully applied to highly selective and efficient glycopeptide enrichment from standard glycoprotein tryptic digest and human serum, indicating the Fe3O4@SiO2@L-Cys nanoparticles are promising materials for glycopeptide analysis in complex biological samples.
2. Material and methods 2.1. Chemicals and materials Iron (Ⅲ) chloride hexahydrate (FeCl3 6H2O), tetraethyl orthosilicate (TEOS), ammonium hydroxide (NH3 H2O, 28 wt%), ethylene glycol, sodium acetate and ammonium bicarbonate (NH4HCO3) were purchased from Shanghai Chemical Corp. Vinyltriethoxysilane (VTES), L-Cysteine, horseradish peroxidase (HRP), bovine serum albumin (BSA) were supplied by Sigma-Aldrich. PNGase F was purchased from New England Biolabs (Ipswich, MA, USA). Human serum was provided by Zhongshan Hospital (Shanghai). Ultrapure water (18.2 MΩ cm) was used for all experiments, purified by a Milli-Q system (Millipore, Bedford, MA). Acetonitrile (ACN) was obtained from Merck (Darmstadt, Germany). All other reagents were of analytical grade. 2.2. Synthesis of Fe3O4 nanoparticles Fe3O4 nanoparticles were synthesized through a hydrothermal method. Specifically, 1.35 g FeCl3 6H2O was dissolved into 75 mL ethylene glycol under magnetic stirring. 3.6 g sodium acetate and 0.2 g anhydrous sodium citrate were added into the mixture, followed by vigorously stirring for 0.5 h. The obtained yellow solution was then transferred to a Teflon-lined stainless steel autoclave. After reacting at 200 °C for 16 h and cooling to room temperature, the obtained Fe3O4 nanoparticles were separated from the solution by a magnet. Then the Fe3O4 nanoparticles were washed with ethanol and deionized water and dried in vacuum at 50 °C overnight. 2.3. Synthesis of Fe3O4@SiO2 nanoparticles Obtained Fe3O4 nanoparticles (0.18 g) were dispersed in a mixed solution containing 160 mL ethanol, 1 mL deionized water and 2 mL NH3 H2O under mechanical stirring for 0.5 h at 30 °C. Subsequently, 1.0 mL tetraethyl orthosilicate (TEOS) was added dropwise within 3 min. The reaction was stirred vigorously at 30 °C for 10 h. After washing with ethanol and deionized water for three times, the resulted Fe3O4@SiO2 nanoparticles were vacuumdried at 50 °C for further use. 2.4. Synthesis of Fe3O4@SiO2@L-Cys nanoparticles The prepared 50 mg Fe3O4@SiO2 nanoparticles were dispersed in 100 mL isopropyl alcohol solution. Then, 2 mL vinyltriethoxysilane (VTES) was added in the mixture under nitrogen
atmosphere for 0.5 h. The mixed solution was mechanical stirred at room temperature for 16 h. The VTES-modified Fe3O4@SiO2 microspheres were collected and washed with ethanol and deionized water repeatedly, and redispersed in ethanol (90 mL) in a dried single-necked flask. The mixture of 50 mg L-Cysteine in water (20 mL) and 2 mg azoisobutyronitrile (AIBN) in ethanol (2 mL) was added in the flask under ultrasonic condition. The resulting mixture was then heated at 60 °C with mechanical stirring for 3 h. The obtained Fe3O4@SiO2@L-Cys microspheres were washed with ethanol and deionized water for three times by magnetic separation and dried in vacuum at 50 °C. 2.5. Characterization Fourier-transform infrared (FT-IR) spectra was obtained on Nicolet Fourier spectrophotometer using KBr pellets (Thermo Fisher, USA). Transmission electron microscopy (TEM) images were collected on JEOL2011 microscope (Japan). Scanning electron microscopy (SEM) images were performed on a Philips XL30 electron microscope (Netherlands). The saturation magnetization curve was monitored by MPMS (SQUID) VSM (Quantum Design, San Diego, USA) at room temperature. Powder X-ray diffraction (XRD) patterns were performed on Bruker D4 X-ray diffractometer with Ni-filtered Cu Kα radiation (40 kV, 40 mA). 2.6. Tryptic digestion of the glycoproteins and human serum The tryptic digestion is a mature and universal methodology approach and has been reported in previous studies [26–28]. 2 mg HRP was dissolved in 1 mL buffer containing 25 mM ammonium bicarbonate (pH 7.9) and denatured in boiling water for 5 min. After reducing in 10 mM DTT at 60 °C for 0.5 h, the protein was alkylated in the dark at 37 °C for 1 h by adding 20 mM IAA. Then the solution was digested with trypsin at an enzyme/protein ratio of 1:40 (w/w) at 37 °C for 16 h. The digest was stored at 20 °C for further use. For tryptic digestion of human serum, the sample was first centrifuged at 12,000 rpm for 10 min. The obtained supernatant (2 μL) was a hundredfold diluted with 25 mM ammonium bicarbonate (pH 7.9) and denatured at 100 °C for 5 min. The mixture was reduced in DTT (10 mM) and then alkylated with IAA (25 mM) in the dark at 37 °C for 1 h. Subsequently, the mixture was digested by trypsin at an enzyme/protein ratio of 1:40 (w/w) at 37 °C for 16 h. The tryptic digestion was lyophilized and stored at 20 °C for further use. 2.7. Enrichment of glycopeptides Fe3O4@SiO2@L-Cys microspheres (150 μg) were washed with loading buffer (90% ACN/H2O, 0.1% TFA) and diluted with 100 μL loading buffer containing 0.4 μg HRP tryptic digestion. The mixture were shaked mildly at 37 °C for 30 min, and then wash with 100 μL loading buffer to remove the non-glycopeptides for three times with the aid of an external magnetic field. The captured glycopeptides were incubated in 10 μL eluting buffer (30% ACN/ H2O, 0.1% TFA) under gentle agitation for 30 min at 37 °C. The elute was detected by MALDI-TOF-MS. For the glycopeptide enrichment from human serum, the lyophilized tryptic digest was dissolved in 200 μL loading buffer. 1 mg Fe3O4@SiO2@L-Cys microspheres were added in the solution and incubated under mild vibration for 30 min at 37 °C. After washing with loading buffer for three times, the mixture were dispersed in eluting buffer under mild vibration for another 30 min at 37 °C. The obtained glycopeptides were lyophilized and redissolved in 25 mM ammonium bicarbonate (pH 7.9). Then, 500 U of PNGase F was added and the mixture was incubated for 12 h at 37 °C to
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remove the glycan moieties. The deglycosylated peptides were evaporated to dryness for further LC-MS analysis. 2.8. Mass spectrometry analysis All the MALDI-TOF-MS experiments were operated in a reflector positive mode on a 5800 Proteomics Analyzer (Applied Biosystems, USA). The Nd/YAG laser was 355 nm, the frequency was 200 Hz and acceleration voltage was 20 kV. The 2, 5-dihydroxybenzoic acid (DHB, 20 mg) was dissolved in 1 mL mixed solution of ACN/H2O/TFA (50:50:0.1, v/v/v) to prepare a matrix. 1 μL sample solution was deposited onto the MALDI plate, followed by dropping 1 μL DHB matrix solution for MS analysis.
3. Results and discussion 3.1. Synthesis and characterization of Fe3O4@SiO2@L-Cys nanoparticles The protocol for the preparation of Fe3O4@SiO2@L-Cys microspheres was illustrated in Scheme 1. Fe3O4 nanoparticles were firstly synthesized via a modified hydrothermal reaction based on a high temperature reduction of Iron(Ⅲ) chloride hexahydrate in the presence of trisodium citrate. Then, a SiO2 layer was formed on the surface of Fe3O4 nanoparticles via sol-gel approach.
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Subsequently, Vinyltriethoxysilane (VTES) was used for the surface modification of Fe3O4@SiO2 nanoparticles to endow abundant carbon-carbon double bonds. Fe3O4@SiO2@L-Cys microspheres were achieved by the thiol-ene click reaction. The thiol-ene click reaction can preserve amino and carboxyl groups in the L-Cysteine, maintaining the superior zwitterionic hydrophilicity. The size and morphology of Fe3O4@SiO2@L-Cys nanoparticles were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). As shown in Fig. 1, the Fe3O4 nanoparticles were uniform in both size and shape, with a mean diameter of about 200 nm. After being coated with SiO2, the size of core-shell Fe3O4@SiO2 nanoparticles was increased to about 240 nm (Fig. 1a). The modification with L-Cysteine on the surface of Fe3O4@SiO2 resulted in an obvious agglomeration, indicating the successful coating of L-Cysteine layer (Fig. 1b). The SEM images of Fe3O4@SiO2 and Fe3O4@SiO2@L-Cys showed spherical shape with little difference in size and morphology (Fig. 1c and d). Energy Dispersive Spectrometer (EDS) analysis of the Fe3O4@SiO2@L-Cys nanoparticles was shown in Table S1. The S element in the L-Cysteine was confirmed, indicating that L-Cysteine was successfully grafted on the surface of silica shell. Fourier transform infrared (FT-IR) spectroscopy was used to confirm the chemical composition of Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2@L-Cys nanoparticles. As shown in Fig. 2a, the peak at 567 cm 1 was assigned to the Fe–O stretching vibration. In the FTIR spectrum of Fe3O4@SiO2 nanoparticles (Fig. 2b), absorption
Scheme 1. Schematic illustrations of the (a) synthetic procedure of the Fe3O4@SiO2@L-Cys nanoparticles and (b) selective glycopeptide enrichment strategy by Fe3O4@SiO2@L-Cys nanoparticles.
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Fig. 1. (a) TEM and (c) SEM images of Fe3O4@SiO2; (b) TEM and (d) SEM images of Fe3O4@SiO2@L-Cys nanoparticles.
Fig. 2. FT-IR spectra of (a) Fe3O4, (b) Fe3O4@SiO2 and (c) Fe3O4@SiO2@L-Cys nanoparticles.
Fig. 3. Magnetic hysteresis curves (c) Fe3O4@SiO2@L-Cys nanoparticles.
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of
(a)
Fe3O4,
(b)
Fe3O4@SiO2
and
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Fig. 4. MALDI-TOF-MS spectra of 4 ng/μL HRP tryptic digest (a) direct analysis; after enrichment by (b) Fe3O4@SiO2@L-Cys, (c) Fe3O4 and (d) Fe3O4@SiO2 nanoparticles. Glycopeptides were marked with numbers in the spectra and detailed information was shown in Table 1.
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Table 1 The detailed information of glycopeptides derived from tryptic digest of HRP after enrichment by Fe3O4@SiO2@L-Cys nanoparticles. Number
m/z
Glycan composition
Peptide sequence
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
2069 2541 2591 2612 2850 3088 3322 3353 3524 3607 3671 3895 4058 4224 4836
PNVSN#IVR SSPN#ATDTIPLVR PTLN#TTYLQTLR MGN#ITPLTGTQGQIR GLIQSDQELFSSPN#ATDTIPLVR GLCPLNGN#LSALVDFDLR QLTPTFYDNSCPN#VSNIVR SFAN#STQTFFNAFVEAMDR GLIQSDQELFSSPN#ATDTIPLVR NQCRGLCPLNGN#LSALVDFDLR GLIQSDQELFSSPN#ATDTIPLVR LHFHDCFVNGCDASILLDN#TTSFR QLTPTFYDNSC(AAVESACPR)PN#VSNIVR-H2O QLTPTFYDNSC(AAVESACPR)PN#VSNIVR LYN#FSNTGLPDPTLN#TTYLQTLR
16
4985
[Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Xyl]1 [HexNAc]1[Fuc]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1 [Hex]3[HexNAc]2[Fuc]1[Xyl]1
LYN#FSNTGLPDPTLN#TTYLQTLR
HexNAc¼N-acetylglucosamine, Hex ¼mannose, Fuc¼ fructose, Xyl¼ xylose. N# denotes the N-linked glycosylation site.
peaks at 1088 and 954 cm 1 were separately attributed to the Si– O–Si and Si–OH stretching vibration. In the FT-IR spectrum of Fe3O4@SiO2@L-Cys nanoparticles (Fig. 2c), new absorption peak at 1640 cm 1 was ascribed to the stretching vibration of C ¼O bond, and the broad peak at around 3300 cm 1 was assigned to the N–H stretching vibration. The X-ray diffraction patterns (XRD) of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@L-Cys nanoparticles were shown in Fig. S1. The characteristic diffraction peaks of Fe3O4 were 30.0°, 35.5°, 42.9°, 57.1° and 62.6°, corresponding to the 2θ values of (220), (311), (400), (511) and (440), respectively. The XRD results were in good agreement with the face-centered cubic phase of Fe3O4 (JCPDS Card no. 19-629), demonstrating that the structure of the nanoparticles remains unchanged after modification. The magnetic properties of the synthesized Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@L-Cys nanoparticles were investigated by a vibrating sample magnetometer (VSM) at room temperature. As shown in Fig. 3, the saturation magnetic (MS) values of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@L-Cys nanoparticles were 81.74, 64.89 and 60.01 emu/g, respectively. Due to superior superparamagnetic properties, Fe3O4@SiO2@L-Cys nanoparticles could be easily separated from solution within several seconds with the help of a magnet. The nanoparticles redispersed quickly in the solution after removing the magnet, which could be applied to the efficient enrichment and separation of glycopeptides. In addition, the hydrophilicity of the core-shell magnetic nanoparticles was evaluated by analyzing the water contact angle using the powder tableting method. As shown in Fig. S2, the water contact angles of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@L-Cys nanoparticles were 12.8°, 22.8° and 22.7°, respectively. The relatively small water contact angles demonstrated the excellent hydrophilicity of as-prepared nanoparticles, indicating promising hydrophilic enrichment of glycopeptides. 3.2. Selective enrichment of glycopeptides by Fe3O4@SiO2@L-Cys nanoparticles As a typical glycoprotein with 9 glycosylation sites, horseradish peroxidase (HRP), has been extensively studied and applied to glycoproteomics analysis [21–23,26,27]. Therefore, HRP tryptic digest was employed as a standard biological sample to evaluate the glycopeptide enrichment efficiency of the synthesized Fe3O4@SiO2@L-Cys nanoparticles. The enrichment strategy
included three steps: incubation for enriching glycopeptides, wash for removing non-glycopeptides and elution for releasing captured glycopeptides. Then, the obtained glycopeptides were detected by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). As shown in Fig. 4a, only one glycopeptide could be observed in direct analysis of 4 ng/μL HRP tryptic digest owing to the severe signal suppression from co-existed non-glycopeptides. However, after enrichment by Fe3O4@SiO2@L-Cys nanoparticles, 16 peaks corresponding to the glycopeptides clearly dominated the spectrum (Fig. 4b), with greatly improved intensity and signal-to-noise (S/N) ratios. Detailed information about the captured glycopeptides was shown in Table 1. Moreover, signals of non-glycopeptides almost disappeared. To verify the hydrophilic enrichment capacity of the material, the efficiency of glycopeptide enrichment were compared by equivalent amount of Fe3O4 and Fe3O4@SiO2 nanoparticles. As the mass spectrum revealed, only 3 glycopeptides was detected with the enrichment of Fe3O4 nanoparticles and 4 peaks of glycopeptides were obtained with the enrichment of Fe3O4@SiO2 nanoparticles (Fig. 4c and d). With excellent hydrophilicity, Fe3O4@SiO2@L-Cys nanoparticles showed better performance in glycopeptide enrichment. The result was attributed to the modification of highly abundant L-Cysteine, which could significantly enhance hydrophilicity of the material with zwitterionic functional groups (–NH2 and –COOH). To evaluate the sensitivity of the glycopeptide enrichment, different concerntration of HRP tryptic digests (2 ng/μL and 0.04 ng/μL) were tested (Fig. 5a and b). As shown in Fig. 5b, when the concerntration of HRP tryptic digest was as low as 0.04 ng/μL, three peaks assigned to glycopeptides were still detected with clean background and high intensity. The selectivity of this method was investigated by the mixture of HRP (standard glycoprotein) and BSA (standard non-glycoprotein) tryptic digest at a mass ratio of 1:50 (HRP: BSA). Due to the serious interference by the non-glycopeptides of BSA tryptic digest, the signals of glycopeptides were hardly detected directly (Fig. 5c). However, as shown in Fig. 5d, 9 glycopeptide peaks with significantly improved intensity appeared after enrichment by Fe3O4@SiO2@L-Cys nanoparticles. The results indicated that the zwitterionic hydrophilic method can enrich glycopeptides with remarkable sensitivity and selectivity. The reproducibility of the Fe3O4@SiO2@L-Cys nanoparticles was also examined using the method. The enriching procedure of
Please cite this article as: R. Wu, et al., Talanta (2016), http://dx.doi.org/10.1016/j.talanta.2016.07.045i
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Fig. 5. MALDI-TOF-MS spectra of (a) 2 ng/μL and (b) 0.04 ng/μL HRP tryptic digest after enrichment by Fe3O4@SiO2@L-Cys nanoparticles. MALDI-TOF-MS spectra of the tryptic digest mixture of HRP and BSA (with a mass ratio of 1:50): (c) direct analysis; (d) after enrichment by Fe3O4@SiO2@L-Cys nanoparticles. Glycopeptides were marked with numbers in the spectra and detailed information was shown in Table 1.
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Fig. 6. MALDI-TOF-MS spectra of 4 ng/μL HRP tryptic digest after enrichment by Fe3O4@SiO2@L-Cys nanoparticles for (a) the third time and (b) the fifth time. Glycopeptides were marked with numbers in the spectra and detailed information was shown in Table 1.
glycopeptides was repeated for five times with the same Fe3O4@SiO2@L-Cys nanoparticles. Before each enriching cycle, the Fe3O4@SiO2@L-Cys nanoparticles were washed sequentially with eluting and loading buffers for several times to remove the adsorbed peptides on the surface of nanoparticles and other impurities. After third and fifth enriching cycle, the obtained signals of glycopeptides were as similar as the first time (Fig. 6), demonstrating the stability and enrichment efficiency of the Fe3O4@SiO2@L-Cys nanoparticles. The practical application of the method was further investigated by detecting glycopeptides and proteins of the human serum sample. The enriching procedure was the same as standard glycopeptide enrichment. The eluted glycopeptides were incubated with PNGase F for deglycosylation. Then the glycosylated peptides were analyzed by LC-MS/MS. A total of 199 unique N-glycosylation sites assigned to 88 glycoproteins were identified (Table S2), indicating the successful application to real biological samples by the method.
4. Conclusion In summary, a novel and efficient method for glycopeptide enrichment was established by fabricating a zwitterionic hydrophilic magnetic core-shell material (Fe3O4@SiO2@L-Cys nanoparticles). With abundant zwitterionic functional groups, L-Cysteine was immobilized based on the thiol-ene click chemistry, distinctly enhancing the hydrophilicity of the material. The Fe3O4@SiO2@L-Cys nanoparticles exhibited excellent performance in HILIC enrichment of glycopeptide, such as high sensitivity, selectivity and reproducibility. Furthermore, the Fe3O4@SiO2@L-Cys nanoparticles were successfully applied to glycopeptide enrichment from human serum sample. In view of these advantages, the innovative method shows a great potential for glycoproteomics analysis in complex biological samples.
Notes The authors declare no competing financial interest.
Please cite this article as: R. Wu, et al., Talanta (2016), http://dx.doi.org/10.1016/j.talanta.2016.07.045i
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Acknowledgement Financial support is gratefully acknowledged from the Research Fund for the Doctoral Program of Higher Education of China (20110071110007, 20100071120053), the National Natural Science Foundation of China (21075022, 20875017, 21105016), the National Basic Research Priorities Program of China (2012CB910602, 2013CB911201), the Shanghai Municipal Natural Science Foundation (11ZR1403200), and Shanghai Leading Academic Discipline Project (B109).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2016.07. 045.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
G. Durand, N. Seta, Clin. Chem. 46 (2000) 795–805. K. Ohtsubo, J.D. Marth, Cell 126 (2006) 855–867. G.W. Hart, R.J. Copeland, Cell 143 (2010) 672–676. M.A. Daniels, K.A. Hogquist, S.C. Jameson, Nat. Immunol. 3 (2002) 903–910. Y. Tian, H. Zhang, Proteom.: Clin. Appl. 4 (2010) 124–132. S.A. Svarovsky, L. Joshi, Anal. Methods 6 (2014) 3918–3936. C. Lei, K. Qian, O. Noonan, A. Nouwens, C. Yu, Nanoscale 5 (2013) 12033–12042. A. Dell, H.R. Morris, Science 291 (2001) 2351–2356. D. Baycin-Hizal, Y. Tian, I. Akan, E. Jacobson, D. Clark, A. Wu, R. Jampol, K. Palter, M. Betenbaugh, H. Zhang, Anal. Chem. 83 (2011) 5296–5303. S. Pan, R. Chen, R. Aebersold, T.A. Brentnall, Mol. Cell. Proteom. 10 (2011) 1–14. Y. Yu, J. Fournier, M. Gilar, J.C. Gebler, Anal. Chem. 79 (2007) 1731–1738. Y. Zhang, B.R. Fonslow, B. Shan, M.C. Baek, J.R. Yates III, Chem. Rev. 113 (2013) 2343–2394. Z. Zhang, D. Sun, Y. Cong, J. Mao, J. Huang, H. Qin, J. Liu, G. Huang, L. Wang, M. Ye, H. Zou, J. Proteom. Res. 14 (2015) 3892–3899. L. Liu, M. Yu, Y. Zhang, C. Wang, H. Lu, ACS Appl. Mater. Interfaces 6 (2014) 7823–7832. Z. Zhang, Z. Sun, J. Zhu, J. Liu, G. Huang, M. Ye, H. Zou, Anal. Chem. 86 (2014) 9830–9837. J. Huang, H. Wan, Y. Yao, J. Li, K. Cheng, J. Mao, J. Chen, Y. Wang, H. Qin, W. Zhang, M. Ye, H. Zou, Anal. Chem. 87 (2015) 10199–10204. Y. Zhang, M. Yu, C. Zhang, Y. Wang, Y. Di, C. Wang, H. Lu, Chem. Commun. 51 (2015) 5982–5985. J.Y. Kim, D. Oh, S.K. Kim, D. Kang, M.H. Moon, Anal. Chem. 86 (2014) 7650–7657. J.A. Ferreira, A.L. Daniel-da-Silva, R.M. Alves, D. Duarte, I. Vieira, L.L. Santos,
469
R. Vitorino, F. Amado, Anal. Chem. 83 (2011) 7035–7043. [20] Z. Tan, H. Yin, S. Nie, Z. Lin, J. Zhu, M.T. Ruffin, M.A. Anderson, D.M. Simeone, D. M. Lubman, J. Proteom. Res. 14 (2015) 1968–1978. [21] X. Zhang, J. Wang, X. He, L. Chen, Y. Zhang, ACS Appl. Mater. Interfaces 7 (2015) 24576–24584. [22] Y. Wang, M. Liu, L. Xie, C. Fang, H. Xiong, H. Lu, Anal. Chem. 86 (2014) 2057–2064. [23] Y. Zhang, W. Ma, D. Li, M. Yu, J. Guo, C. Wang, Small 10 (2014) 1379–1386. [24] M.R. Larsen, S.S. Jensen, L.A. Jakobsen, N.H. Heegaard, Mol. Cell. Proteom. 6 (2007) 1778–1787. [25] T. Jin, Z. Xiong, X. Zhu, N. Mehio, Y. Chen, J. Hu, W. Zhang, H. Zou, H. Liu, S. Dai, ACS Macro Lett. 4 (2015) 570–574. [26] C. Bi, Y. Zhao, L. Shen, K. Zhang, X. He, L. Chen, Y. Zhang, ACS Appl. Mater. Interfaces 7 (2015) 24670–24678. [27] B. Jiang, Q. Wu, N. Deng, Y. Chen, L. Zhang, Z. Liang, Y. Zhang, Nanoscale 8 (2016) 4894–4897. [28] J. Li, F. Wang, J. Liu, Z. Xiong, G. Huang, H. Wan, Z. Liu, K. Cheng, H. Zou, Chem. Commun. 51 (2015) 4093–4096. [29] H. Lin, J. Ou, Z. Zhang, J. Dong, M. Wu, H. Zou, Anal. Chem. 84 (2012) 2721–2728. [30] L. Yu, X. Li, Z. Guo, X. Zhang, X. Liang, Chem. Eur. J. 15 (2009) 12618–12626. [31] X. Zou, J. Jie, B. Yang, Chem. Commun. 52 (2016) 3251–3253. [32] J. Zheng, Y. Xiao, L. Wang, Z. Lin, H. Yang, L. Zhang, G. Chen, J. Chromatogr. A 1358 (2014) 29–38. [33] Y. Zhao, L. Yu, Z. Guo, X. Li, X. Liang, Anal. Bioanal. Chem. 399 (2011) 3359–3365. [34] Y.W. Zhang, Z. Li, Q. Zhao, Y.L. Zhou, H.W. Liu, X.X. Zhang, Chem. Commun. 50 (2014) 11504–11506. [35] M.H.J. Selman, M. Hemayatkar, A.M. Deelder, M. Wuhrer, Anal. Chem. 83 (2011) 2492–2499. [36] P. Dedvisitsakul, S. Jacobsen, B. Svensson, J. Bunkenborg, C. Finnie, P. Hagglund, J. Proteom. Res. 13 (2014) 2696–2703. [37] Z. Xiong, L. Zhao, F. Wang, J. Zhu, H. Qin, R. Wu, W. Zhang, H. Zou, Chem. Commun. 48 (2012) 8138–8140. [38] Y. Chen, Z. Xiong, L. Zhang, J. Zhao, Q. Zhang, L. Peng, W. Zhang, M. Ye, H. Zou, Nanoscale 7 (2015) 3100–3108. [39] M. Pabst, I. Benesova, S.R. Fagerer, M. Jacobsen, K. Eyer, G. Schmidt, R. Steinhoff, J. Krismer, F. Wahl, J. Preisler, R. Zenobi, J. Proteom. Res. 15 (2016) 326–331. [40] Y. Deng, Y. Cai, Z. Sun, J. Liu, C. Liu, J. Wei, W. Li, C. Liu, Y. Wang, D. Zhao, J. Am. Chem. Soc. 132 (2010) 8466–8473. [41] X. Xu, C. Deng, M. Gao, W. Yu, P. Yang, X. Zhang, Adv. Mater. 18 (2006) 3289–3293. [42] D. Yu, A. Shen, Z. Guo, Y. Yan, J. Yan, G. Jin, X. Liang, Chem. Commun. 51 (2015) 14778–14780. [43] I. Tijunelyte, E. Guenin, N. Lidgi-Guigui, F. Colas, J. Ibrahim, T. Toury, M. Lamy de la Chapelle, Nanoscale 8 (2016) 7105–7112. [44] B. Jiang, Y. Liang, Q. Wu, H. Jiang, K. Yang, L. Zhang, Z. Liang, X. Peng, Y. Zhang, Nanoscale 6 (2014) 5616–5619. [45] E. Ataman, C. Isvoranu, J.N. Andersen, J. Schnadt, K. Schulte, J. Phys. Chem. Lett. 2 (2011) 1677–1681. [46] Y. Liang, C. Wu, Q. Zhao, Q. Wu, B. Jiang, Y. Weng, Z. Liang, L. Zhang, Y. Zhang, Anal. Chim. Acta 900 (2015) 83–89.
Please cite this article as: R. Wu, et al., Talanta (2016), http://dx.doi.org/10.1016/j.talanta.2016.07.045i