Article pubs.acs.org/ac
Highly Selective Enrichment of N‑Linked Glycan by CarbonFunctionalized Ordered Graphene/Mesoporous Silica Composites Nianrong Sun,† Chunhui Deng,*,† Yan Li,*,‡ and Xiangmin Zhang† †
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203, China
‡
S Supporting Information *
ABSTRACT: Abnormal protein glycosylation has been demonstrated to be associated with many diseases; therefore, it is very important to conduct a comprehensive structure analysis of glycan for prognosis and diagnosis of diseases, such as cancer. In this work, for the first time, carbon-functionalized ordered graphene/mesoporous silica composites (denoted as C-graphene@mSiO2) with large surface area and uniform pore size were designed and synthesized. By taking advantage of the special interaction between the carbon and glycans as well as size-exclusion ability, 25 N-linked glycans released from ovalbumin were observed clearly with strong MS signals and increased signal-to-noise (S/N) ratio. In addition, after enrichment with the C-graphene@ mSiO2 composites, 48 N-linked glycans (S/N > 10) with sufficient peak intensities were obtained from only 400 nL of healthy pristine human serum. The facile and low-cost synthesis method as well as high selective enrichment ability of the novel Cgraphene@mSiO2 composite makes it a promising tool for glycosylation research.
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Therefore, it is very urgent in glycosylation research to develop novel methods for highly selective enrichment of glycans in biosamples. Up to now, many methodologies have been used to enrich glycan from biological samples. Several methods including organic solvent precipitation,7 lection,8 and hydrophilic interaction chromatography (HILIC)9 have been used in the enrichment of N-glycan. HILIC-based approaches with high selectivity and good reproducibility, which rely upon the physical interaction between the stationary phase and the glycan, are the most widespread applications for glycan analysis. Unfortunately, HILIC adsorbents, which are prepared by chemical modification of the monolayer molecules on the surface, lead to relatively low enrichment capacity. Recently, ordered mesoporous silica materials, such as MCM-41, have emerged as promising alternatives for the selective enrichment of peptides and glycans from complex biosamples due to their relatively large surface area, high stability, and uniform
lycosylation, which is an important and ubiquitous protein post-translational modification, is a process where glycan is attached to proteins or lipids under the control of enzymes and is involved in numerous physiological and pathological processes, including cell growth, cell adhesion, cell−cell recognition, etc.1,2 Generally, protein glycosylation can be classified into two main categories: the glycans of Nlinked glycosylation which are conjugated to proteins through asparagine residues consisting of a consensus tripeptide sequence of Asn-X-Ser/Thr (X can be any amino acid except proline), and the glycans of O-glycosylation which are linked to serine or threonine residues. Interestingly, N-linked glycans were released from proteins enzymatically with peptide-Nglycosidases (PNGases),3 while no enzyme has been found to be able to release O-linked glycans yet. Besides, the altered structures of glycan, in particular N-linked glycan, have been confirmed to be bound in various diseases,4−6 such as cancer. Mass spectrometry (MS) is the most widely used analytical technique in glycan profiling for glycosylation research. However, glycan signals from complex biological samples are hardly detected by direct MS analysis due to interference from residual proteins and the low abundance of the available glycan. © 2014 American Chemical Society
Received: December 18, 2013 Accepted: January 24, 2014 Published: January 24, 2014 2246
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mesoporous SiO2 layer was directly coated on the surface of the acid-treated graphene by utilizing tetraethyl orthosilicate (TEOS) as a silicon precursor, and CTAB as the structuredirecting agent. Finally, C-graphene@mSiO2 composites were obtained by pretreating the graphene@mSiO2−CTAB composites with sulfuric acid and further carbonizing under the protection of nitrogen. Importantly, CTAB is prone to degrade under high temperature, which would result in a decrease in the carbonization rate of CTAB, so it is necessary to pretreat the graphene@mSiO2−CTAB composites before calcination. According to Hyson’s reports,24 ordered mesoporous carbon materials can be synthesized by directly carbonizing silica/ triblock copolymer nanocomposites which are pretreated with sulfuric acid, the role-playing catalyst in the process of carbonization and dehydration. Therefore, in this study, to prevent the degradation of CTAB and thus increase the content of carbon in the final product, the as-synthesized graphene@ mSiO2−CTAB composites were also pretreated using sulfuric acid at a lower temperature before carbonization and then successfully carbonized at elevated temperature in a N2 atmosphere. According to a previous report,16 873 K was chosen as the calcination temperature under nitrogen flow to acquire the final product. Scanning electron microscopy (SEM) shows that Cgraphene@mSiO2 composites have a smooth surface and maintain the structure of graphene (Figure 1a). Transmission electron microscopy (TEM) indicates that the composites possess well-organized pore channels (Figure 1b).
mesochannels, which can extract low molecular weight targets while excluding proteins with higher molecular weight.10−12 Many innovative methods based on mesoporous materials have been established for the selective enrichment of endogenous peptides,13−17 N-glycans,18,19 and phosphopeptides.20 In our previous reports, functionalized mesoporous silica materials were synthesized for peptidome research.13,14,16 It has been shown that the increase in surface area is significant for enhancing the enrichment ability of mesoporous silica for complex biological samples.13−20 Hence, the design and synthesis of mesoporous materials with high surface area for glycan and peptide enrichment have triggered increasing academic proteome research. Graphene has attracted increasing research interest since its discovery in 2004. To date, graphene has been widely applied in diverse fields such as nanoelectronic devices, transistors, and gene sequencing. Recently, on the basis of high surface area, graphene-based mesoporous silica materials have been prepared for peptide and glycan enrichment for proteomics research.13,14,21 In this study, for the first time, carbon-functionalized ordered graphene/mesoporous silica composites (denoted as Cgraphene@mSiO2) with short mesoporous channels and high surface area were directly prepared by using graphene as the support and a structure-directing agent as the carbon source. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was not only used as the structure-directing agent during the formation of the mesoporous silica layer on the graphene support but also directly transited to carbon in the carbonization step under an atmosphere of nitrogen. The asprepared composite possessed several merits such as high content of carbon which can enrich glycan via hydrophilic and polar interactions,22,23 highly ordered mesopores with suitable size which can exclude large proteins, and high surface area which can enhance the enrichment efficiency. By taking advantage of these merits, the novel composites were utilized to enrich N-linked glycans from complex biological samples with high selectivity and efficiency.
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RESULTS AND DISCUSSION Synthesis and Characterization of C-graphene@ mSiO2. The synthetic approach for C-graphene@mSiO 2 composites is shown in Scheme 1. Briefly, at first, the primitive graphene slices were treated with concentrated nitric acid which enabled the surface of graphene to fill with hydrophilic groups (such as hydroxy and carboxyl) for the next step. Then a
Figure 1. SEM images (a) and TEM images (b) of the C-graphene@ mSiO2 nanocomposites.
In addition, the morpha and mesoporous structure of the Cgraphene@mSiO2 composites and H2SO4-pretreated graphene@mSiO2−CTAB composites were characterized by Xray powder diffraction (XRD). An inconspicuous diffraction around 2° was noticed (Figure S1a, Supporting Information), which can be interpreted as the loss of a small percentage of CTAB in the process of washing the materials. A well-resolved diffraction peak with low-angle XRD patterns corresponding to (100) indicated the existence of a two-dimensional hexagonal ordered mesoporous structure (Figure S1b, Supporting Information); namely, the CTAB was carbonized successfully so that mesoporous structure was formed. A strong diffraction peak at around 26° in the wide-angle XRD pattern of each structure could be due to a multitude of graphitic layers in the nanocomposites (Figure S2, Supporting Information), indicative of the retained structure of graphene after high temperature carbonization. Also, the N2 sorption isotherm indicates the existence of interstitial structures in the C-graphene@mSiO2
Scheme 1. Synthesis Route to Carbon-Functionalized Ordered Graphene/Mesoporous Silica Nanocomposites
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Figure 2. MALDI-TOF MS analysis of N-linked glycan released from OVA: (a) without enrichment; (b) after enrichment by active carbon materials; (c) after enrichment by C-graphene@mSiO2 nanocomposites (the glycan structures were searched by using http://www. functionalglycomics.org based on the mol wt of the oligosaccharide: green, mannose; yellow, galactose; blue, GlcNAc).
noise ratio (S/N) of 37.15. According to Zou’s method,16,17 the calculated enrichment yield is about 190 mg of β-CD per gram of C-graphene-@mSiO2 composites (Figure S7, Supporting Information), indicating a large enrichment capacity for glycans. To further investigate the enrichment ability for glycans, the C-graphene@mSiO2 composites were applied to enrich the Nlinked glycan in the digestion solution of ovalbumin. Active carbon materials were also used to extract N-linked glycan for comparison. Figure 2shows that no glycan was detected before the enrichment (Figure 2a). After extraction with active carbon, a total of 24 N-linked glycans were detected (Figure 2b), indicating the ability of carbon for trapping N-linked glycans from the protein digest. After the enrichment with Cgraphene@mSiO2 composites, 25 N-linked glycans with much stronger MS signals were detected (Figure 2c), exhibiting better enrichment efficiency than that achieved with active carbon materials. Besides the large surface area, the C-graphene@mSiO2 composites also have ordered mesoporous channels. Therefore, these novel materials can selectively enrich glycan while excluding proteins in real samples. To evaluate the sizeexclusion ability of C-graphene@mSiO2 composites, a more complex sample containing ovalbumin digests with different amounts of BSA as the interference protein was used. Without enrichment, only nine N-linked glycans were detected when the ratio of BSA to OVA was 10:1 (Figure 3c), while strong MS signals of BSA were identified, as shown in the inset MS spectrum of Figure 3c. When the ratio was increased to 100:1,
composites (Figure S3, Supporting Information), and the aperture distribution curve reveals a uniform mesopore of about 2.8 nm. It is estimated that the nanocomposites have a large Brunauer−Emmett−Teller (BET) surface area of 372 m2/g and a total pore volume of 0.42 cm3/g. As characterized by Raman spectroscopy (Figure S4, Supporting Information), three sharp peaks around 1330 cm−1, 1574 cm−1, and 2674 cm−1 were observed, which were assigned to the characteristic peaks of graphene. Simultaneously, the slight difference around 1330 cm−1 between Figure S4a and Figure S4b is due to the presence of amorphous carbon, which further demonstrates the successful carbonization of CTAB. Furthermore, elemental analysis was adopted for the sake of assessing the content of carbon in the nanocomposite products. Figure S5 (Supporting Information) shows that there is no large difference between the contents of the four elements, respectively, indicating the successful carbonization of CTAB inside the mesoporous pore. All results indicate that the designed C-graphene@mSiO2 composites with high surface area were successfully synthesized. Application of C-graphene@mSiO 2 in N-Linked Glycan Enrichment. To evaluate the glycan enrichment efficiency of the C-graphene-@mSiO2 composites, β-cyclodextrin (β-CD, seven-sugar-ring cyclic oligosaccharide) was selected as the standard glycan. As revealed by mass spectral analysis, an aqueous solution of β-CD at a concentration of 0.5 fmol/μL was difficult to be detected (Figure S6a, Supporting Information). However, after the enrichment with the Cgraphene-@mSiO2 composites (Figure S6b, Supporting Information), the peak of β-CD was observed with a signal-to2248
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Figure 3. . MALDI-TOF MS analysis of N-glycans released from OVA digests of BSA (w/w) at 1:0, 1:10, and 1:100 (a, c, e) before and (b, d, f) after enrichment with C-graphene@mSiO2 nanocomposites. The peaks marked with asterisks represent glycans released from OVA. Peaks marked with red stars are the signals of N-linked glycans.
thoroughly purify the N-linked glycans that were adsorbed on the C-graphene@mSiO2 composites, the elution was repeated several times until no N-linked glycans were observed in the eluent with MALDI-TOF MS (inset, FigureS9, Supporting Information). Then the C-graphene@mSiO2 composites were washed with deionized water three times to recycle. The regenerated C-graphene@mSiO2 composites were used to enrich N-linked glycans from OVA digests three consecutive times. Although the MS signals of N-linked glycans slightly decreased, 25 N-linked glycans can still be identified with the regenerated C-graphene@mSiO2 composites in the third use (Figure S9b, Supporting Information). The enriched glycan amount after the third application and the first application was compared, and the percentage value varied from 87.6% to 107.8% (Table S1, Supporting Information). The above results suggest that the C-graphene@mSiO2 nanocomposites are fairly stable and can be reused for capturing N-linked glycan. There are close connections between glycan in human serum and many diseases;25 therefore, it is of great scientific significance to research the selective enrichment and analysis of glycan in human serum. Inspired by the distinct performance of C-graphene@mSiO2 composites with high surface area, size-
no glycan signals were observed (Figure 3e), which might be due to strong interference from the protein. In comparison, after being enriched with C-graphene@mSiO2 composites, the peak intensities of N-linked glycans maintained a fairly stable level despite the increase in the BSA/OVA ratio from 0:1 to 100:1. Even when the BSA to OVA ratio was increased to 100:1, 21 N-linked glycans (S/N > 10) with obviously increased MS signals could still be detected (Figure 3f). Moreover, no protein signals were found in the MS spectrum after enrichment (inset, Figure 3d). For comparison, active carbon was also used to treat the same complex sample containing ovalbumin digests with different amounts of BSA. The peak intensities of N-linked glycans decreased significantly with the increase in the ratio of BSA/OVA (Figure S8, Supporting Information). The above results indicate that highly efficient enrichment of glycans were obtained with Cgraphene@mSiO2 composites that combine high retention on the carbon surface with the size-exclusion effect of the mesopores. The reusability of C-graphene@mSiO2 composites was also tested through a relative quantitative method with maltoheptaose (m/z = 1152.0) added as the internal standard. To 2249
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exclusion to protein, and specific adsorption, human serum was utilized to further corroborate the effectiveness and selectivity of the C-graphene@mSiO2 composites for the enrichment of N-linked glycan released from complex real samples. It is necessary to prevent interference from endogenous peptides by their removal using an ultrafilter (UF) before the PNGase F digestion. Encouragingly, after enrichment with the Cgraphene@mSiO2 composites, 48 N-linked glycans (S/N > 10) with significantly increased S/N ratio were obtained from only 400 nL of pristine human serum. The various structures of N-glycans are displayed in Table S2, Supporting Information. All of the above results suggest that the C-graphene@mSiO2 composites have high potential for N-glycan enrichment from complex biosamples.
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CONCLUSIONS In this work, carbon-functionalized ordered graphene/mesoporous silica was successfully designed. The as-made composites possess large surface area (372 m2/g), highly ordered mesopores with uniform pore size (2.8 nm), and high content of carbon. On the basis of the specific interaction between the carbon and glycans as well as the size-exclusion ability, the composites exhibit good performance in the selective capture of N-linked glycans in protein digests and human serum. The facile and low-cost synthesis method as well as high selective enrichment ability of the novel C-graphene@ mSiO2 composite makes it a promising tool for glycosylation research.
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ASSOCIATED CONTENT
S Supporting Information *
Additional data for C-graphene@mSiO2 composites characterization and their performance in N-linked glycan enrichment. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Fax: +86-21-65641740. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Basic Research Priorities Program (2012CB910602, 2013CB911201), the National Natural Science Foundation of China (21075022, 20875017, 21105016), Research Fund for the Doctoral Program of Higher Education of China (20110071110007, 20100071120053), Shanghai Municipal Natural Science Foundation (11ZR1403200), and Shanghai Leading Academic Discipline Project (B109).
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