Magnetic nanoporous hybrid carbon from core–shell metal–organic frameworks for glycan extraction

Abstract

Magnetic nanoporous carbon (NPC) materials, which can be thoroughly separated from an aqueous solution easily, are very promising adsorbents. In this study, magnetic nanoporous hybrid carbon materials were synthesized as a novel enrichment platform for glycan analysis using core–shell metal–organic frameworks (MOFs) as a sacrificial template and carbon precursor. The designed and synthesized magnetic nanoporous hybrid carbon materials possessed hybrid merits, including high surface area, cut-off effect of uniform pore size, high graphitic carbon content and strong magnetic response. Considering the specific interaction between graphitized carbons and glycan, the magnetic nanoporous hybrid carbon materials with good repeatability were successfully used to enrich N-glycans from both standard protein digests and human serum.

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Introduction

Glycosylation is an important post-translational modification (PTM) and generates more proteomic multiplicity than any other PTM. In eukaryotes, glycans are conjugated to Asn residues (N-glycans), which present in the tripeptide consensus sequence of Asn-Xaa-Ser/Thr (Xaa could be any amino acid except Pro) or Ser or Thr residues (O-glycans). These forms of glycosylation are implicated in a wealth of biological events, including intracellular sorting, uptake, secretion, cell–cell communication, as well as protein folding and trafficking^1–3 and are therefore of great biological interest. Dynamic changes in glycosylation have been demonstrated to accompany disease states, e.g., Alzheimer's and cancer progression,^4,5 indicating that specific glycosylation forms may serve as disease biomarkers or therapeutic targets. Building connections between glycan functions and their structures, monitoring the variation of glycan structure in disease diagnosis and prognosis, and elucidating pathological mechanisms^6–8 are significant research fields. To achieve these goals, developing methodologies with high-throughput, high stability, high accuracy and sensitivity for glycan analysis are critical. Mass spectrometry (MS) is an often chosen method to profile the proteome. However, it is uniquely problematic to directly profile glycan by MS because of their low abundance and poor ionization efficiency. Therefore, selective enrichment of glycan from bio-samples is particularly significant. To date, many methods have been developed for glycan enrichment, such as organic solvent precipitation, lection, and hydrophilic interaction chromatography (HILIC) and mesoporous carbon materials.^9–14 It has been demonstrated that the utilization of carbon materials for glycan enrichment for glycosylation research is a very important strategy. Preparation of materials with high surface area and large amounts of carbon for glycosylation analysis has become one of the research hotspots.

Metal–organic frameworks (MOFs) are an emerging class of very promising crystallized porous polymeric materials self-assembled straightforwardly by the coordination of metal ions/clusters and organic linkers. Because MOFs possess various properties, such as good thermal stability, diverse structures and morphologies, incredibly high internal surface areas and ultra-high porosity,^15–18 they are gaining increasing attention due to their potential analytical and biological applications.^19–21 Recently, the molecular sieving effect of MOFs has been studied for the selective extraction of low-abundance peptides from complex biosamples with high-abundance proteins excluded before mass spectrometry analysis.^22–24 More recently, MOF materials were also successfully applied to the selective enrichment of phosphopeptides for phosphoproteome research.²⁵ However, to the best of our knowledge, there are no reports on the application of MOFs and the materials derived from MOFs for glycosylation research to date.

Recently, a novel strategy has been successfully developed for the preparation of nanoporous carbon materials via the high-temperature treatment of MOFs.^26–31 MOFs serving as a sacrificial template and carbon precursor simplify the synthetic process. The NPCs from MOFs have been applied as electrode materials for supercapacitors and adsorbents for water treatment, etc.^32,33 In general, NPCs derived from ZIF-8 crystals possess a relatively large surface area (1499 m² g⁻¹), but the carbon presents an amorphous state. On the contrary, NPCs gained from ZIF-67 crystals have a high graphitic carbon content, while the surface area is relatively lower than ZIF-8 because the high porosity of NPCs are easily sacrificed during the process of graphitization. To realize both a high graphitic structure and high surface area, the magnetic nanoporous hybrid carbon materials (denoted as Mag-NHCs) were synthesized from core–shell ZIF-8@ZIF-67 crystals via high-temperature treatment in this study. The feasibility of the material for selective enrichment of glycans was also investigated.

Results and discussion

The preparation and characteristics of Mag-NHCs

The procedure used for the preparation of Mag-NHCs is shown in Fig. 1.

Fig. 1 Schematic of the procedure used for Mag-NHCs preparation and glycan enrichment.

First, the Zn-based zeolitic imidazolate frameworks, [Zn(mim)₂]_n (ZIF-8; mim = 2-methylimidazole), were synthesized as the core. Then, the Co-based zeolitic imidazolate frameworks, [Co (mim)₂]_n (ZIF-67) were then coated on the outside of the ZIF-8 crystals as the shell via a seed-mediated growth technique. Subsequently, Mag-NHCs were obtained via the high temperature treatment of the ZIF-8@ZIF-67 crystals under the protection of a nitrogen atmosphere (Fig. 1). From Fig. S1A and S2A,† scanning electron microscopy (SEM) images and transmission electron microscopy (TEM) images of the ZIF-8 crystals show uniformly dispersed rhombic dodecahedral shapes, which are beneficial for coating uniform ZIF-67 crystals. The Co²⁺ ions on the surface of the ZIF-8 core were immobilized owing to the coordinative interaction with 2-methylimidazole units and then the epitaxial growth of the ZIF-67 shell was attributed to the extra additive 2-methylimidazole units.

As observed in Fig. S1B and S2B,† the structure and morphology of the ZIF-8@ZIF-67 crystals were almost the same as the ZIF-8 crystals, with only slight size-changes. The wide angle X-ray diffraction (XRD) pattern of the ZIF-8 crystals and ZIF-8@ZIF-67 crystals gave a better illustration of the core–shell structure (Fig. S3†).

As revealed by the SEM image (Fig. 2A) and TEM images (Fig. 2B), the as-prepared Mag-NHCs retained the original rhombic dodecahedron shapes with a distorted and bumpy surface. Fig. 2C (including the inset) show the Mag-NHCs possessed a highly porous structure. To further explore the porosity of the Mag-NHCs, a N₂ adsorption–desorption measurement was conducted at 77 K. As observed in Fig. 3, it could be estimated that the average pore size of the Mag-NHCs was about 3.52 nm. The calculated Brunauer–Emmett–Teller (BET) surface area and total pore volume were about 355.91 m² g⁻¹ and 0.32 cm³ g⁻¹, respectively. When compared with a previous report,²⁶ the BET surface area of the NPCs obtained from ZIF-67 crystals was calculated to be 211 m² g⁻¹, which was less than that of the core–shell Mag-NHCs prepared in this study. In addition, as shown in Fig. S4,† the Mag-NHCs can be dispersed in water and magnetically separated.

Fig. 2 SEM image (A), TEM images (B and C) and Raman spectrum (D) of the Mag-NHCs. The inset in (C) shows the detailed porous structure.

Fig. 3 The N₂ adsorption–desorption isotherm of the Mag-NHCs. The inset shows the pore size distribution.

The wide-angle XRD pattern of the Mag-NHCs exhibited a broad diffraction peak at around 26° belonging to the typical (002) diffraction of graphitic carbon (Fig. S5†), indicating the successful formation of graphite-type carbon. According to a previous report, transition metals can act as a catalyst for promoting the graphitization of carbon.^34,35 The degree of graphitization was determined by the chosen carbonization temperature. The obvious 2D band in the Raman spectrum of the Mag-NHCs (Fig. 2D) implied that the graphitic carbon structure was well developed in the core–shell porous materials, which should be attributed to the existence of Co particles and the applied carbonization temperature. However, the Raman spectra of the ZIF-8 crystals and ZIF-8@ZIF-67 crystals were totally different from the Raman spectrum of Mag-NHCs, as observed in the Fig. S6;† there was no characteristic peak of graphitic carbon. In addition, the content of carbon in the Mag-NHCs was assessed by elemental analysis (Fig. S7†); the percentage of C content estimated from energy dispersive X-ray (EDX) detector was 83.83 atomic%, implying a high carbon content. All the abovementioned results indicated that the Mag-NHCs designed with intriguing features were successfully synthesized and anticipated to be an ideal candidate for glycan analysis.

Application of the Mag-NHCs in N-glycans enrichment

With the narrow pore size distribution of the Mag-NHCs (pore size, ca. 3.52 nm), the cut-off ability of the Mag-NHCs to protein was evaluated through capturing glycan from a complex mixture containing a number of proteins (non-glycoprotein BSA and glycoprotein OVA) and the PNGase F digested glycoprotein (OVA enzymatic hydrolysis solution). The MALDI mass spectra acquired from direct analysis and after Mag-NHCs enrichment of the complex mixture are displayed in Fig. 4. Without any treatment, the MS signals of N-glycan released from OVA were hardly recognized and only four N-glycans were observed (Fig. 4A, OVA digests : BSA : OVA = 1 : 0 : 0), presumably because of the interference of residual protein in the OVA digests (the inserted MS spectrum in Fig. 4A). Along with the increase in protein proportion (Fig. 4C, OVA digests : BSA : OVA = 1 : 50 : 50 and Fig. 4E, OVA digests : BSA : OVA = 1 : 100 : 100) no signals of N-glycan were detected, while strong MS signals of protein (both BSA and OVA) were found in the illustration shown in Fig. 4C and E. On the contrary, after being enriched by the Mag-NHCs, over twenty N-glycans with fairly stable peak intensities were easily identified, even when heavily interferential protein was utilized (Fig. 4B, D and F; the detailed glycan structure information is listed in Table S1†). Furthermore, no signals of protein residues were detected in the MALDI mass spectra after enrichment (inset, Fig. 4D and F). These results indicate that the uniform mesopores of the Mag-NHCs have a promising size-exclusive effect to proteins for glycan analysis. In addition, over twenty N-glycans with clear backgrounds can be identified after treatment with the Mag-NHCs three consecutive times, indicating a good repeatability (Fig. S8†). In addition, the limit of detection (LOD) was estimated to be about 2 ng μL⁻¹ (Fig. S9†). The abovementioned results implied that the Mag-NHCs designed possessed a highly efficient enrichment capacity by taking advantage of the size-exclusion effect of the mesopores and the strong retention on the large carbon surface.

Fig. 4 MALDI mass spectra of N-glycans from ovalbumin digest containing OVA and BSA with different ratios (w/w). (A), (C) and (E) are MALDI mass spectra for samples without any treatment; (B), (D) and (F) are MALDI mass spectra for samples after Mag-NHCs enrichment, with the ratio of ovalbumin digests/OVA/BSA at 1 : 0 : 0 (A and B), 1 : 50 : 50 (C and D) and 1 : 100 : 100 (E and F), respectively.

Furthermore, the enrichment recovery of the Mag-NHCs was roughly investigated through several parallel experiments with exactly the same steps. Namely, nine identical aqueous samples consisting of 500 ng of maltoheptaose were treated with the same amount of Mag-NHCs prior to MS analysis. The results are displayed in Fig. S10 and Table S2.† The recovery of the Mag-NHCs was calculated to exceed 82%. All the abovementioned results indicated that the Mag-NHCs designed were promising candidates for glycan analysis.

Human serum, the common clinical specimen, is considered to be of great importance in disease diagnosis and therapies. To date, tremendous efforts have been devoted to find serum biomarkers. However, because the dynamic range of serum protein concentrations is more than 10 orders of magnitude, exploring and developing novel analytical methods and technologies for serum proteins, as well as glycans in glycoprotein is a challenging task. The enrichment capacity of the Mag-NHCs was further investigated by extracting N-glycans released from human serum. An ultra-filter (UF) was employed to remove the interference of endogenous peptides from human serum before the PNGase F treatment. Encouragingly, 38 N-glycans (signal-to-noise, S/N > 10) were identified from human serum (Fig. S11†). For comparison, both active carbon and NPCs from ZIF-67 crystals were used to capture N-linked glycans from human serum with 23 and 27 N-linked glycans identified, respectively. The obtained results indicate the application of Mag-NHCs for N-glycan enrichment is a promising approach for glycan analysis in complex biosamples.

Conclusions

In conclusion, we have designed and synthesized magnetic nanoporous hybrid carbon materials (denoted as Mag-NHCs) via the high temperature treatment of core–shell MOFs. By taking advantage of the high surface area, cut-off effect of uniform pore size, high graphitic carbon content and the specific interaction between graphitized carbons with glycan, the Mag-NHCs were successfully applied to enrich N-glycans from simulated samples and human serum samples. Over twenty N-glycans could be captured with good repeatability by employing OVA as the standard glycoprotein and the LOD was estimated to be about 2 ng μL⁻¹. It can be concluded that the employment of Mag-NHCs for N-glycan extraction will be a feasible and potential approach for glycan analysis by MALDI-TOF-MS, which is a promising candidate for glycan analysis in early disease diagnosis.

Acknowledgements

This study was supported by the National Basic Research Priorities Program (2012CB910602, 2013CB911201), the National Natural Science Foundation of China (21425518, 21075022, and 21275033), and Research Fund for the Doctoral Program of Higher Education of China (20100071120053).

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Footnote

† Electronic supplementary information (ESI) available: Characterization images of the material and identified data of N-glycans. See DOI: 10.1039/c6ra01434h

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