Elsevier

Analytica Chimica Acta

Volume 970, 1 June 2017, Pages 47-56
Analytica Chimica Acta

Ultrathin Au nanowires assisted magnetic graphene-silica ZIC-HILIC composites for highly specific enrichment of N-linked glycopeptides

https://doi.org/10.1016/j.aca.2017.03.014Get rights and content

Highlights

  • L-Cys functionalized magnetic graphene oxide was conveniently preapred for efficient enrichment of glycopeptides.

  • The ultrathin Au nanowires provided numerous of reaction sites and were easily grafted with an abundant amount of L-Cys for the enrichment of glycopeptides.

  • The prepared composites exhibited superior performance in glycopeptide enrichment.

  • The proposed method possesses great application potential in glycopeptide enrichment for complex biological samples enrichment.

Abstract

Protein glycosylation has been proven to participate in a variety of complex biological processes; however, the low abundance of glycopeptides in natural samples makes it essential to develop methods to isolate and enrich glycopeptides. In this study, a novel ultrathin Au nanowire assisted zwitterionic hydrophilic magnetic graphene oxide (GO-Fe3O4/SiO2/AuNWs/L-Cys) was synthesized with the good biocompatibility of GO, strong magnetic responses of Fe3O4, large surface area of ultrathin Au nanowires and excellent hydrophilicity of L-Cys via four simple and rapid steps. The ultrathin Au nanowires have a one-dimensional structure and were easily grafted with an abundant amount of L-Cys for the enrichment of glycopeptides. After the GO-Fe3O4/SiO2/AuNWs/L-Cys composites were applied to glycopeptide enrichment, 26 glycopeptides from a human IgG digest could be identified, with a detection limit as low as 10 fmol. Due to the abundant amount of grafted L-Cys, the composites also showed a large binding capacity (150 μg mg−1). Furthermore, the composites were applied for the analysis of real biological samples. A total of 793 glycopeptides from 467 glycoproteins were identified in three replicate analyses of 40 μg of mouse liver proteins. The results demonstrated the great potential of GO-Fe3O4/SiO2/AuNWs/L-Cys composites for the analysis of glycoproteins.

Graphical abstract

Synthetic process of GO-Fe3O4/SiO2/AuNWs/L-Cys and analysis of glycopeptides using GO-Fe3O4/SiO2/AuNWs/L-Cys.

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Introduction

As one of the most complicated and important post-translational modifications, protein glycosylation has been proven to participate in a variety of complex biological processes, such as molecular recognition, cell signal transduction, and protein folding [1], [2], [3], [4]. A number of studies have revealed that many human diseases are related to abnormal N-glycosylation [5]. Therefore, characterization of glycoprotein structures and identification of glycosylation sites are significant for the study of glycoprotein functions and pathology. To date, the commonly used methods to characterize glycoproteins have been based on mass spectrometry (MS) techniques [6], [7]. However, the low abundance of glycosylated peptides and the co-existence of non-glycopeptides and salts lead to target signal suppression, which makes the direct MS analysis of glycopeptides a challenge [8].

Thus, several highly efficient and selective enrichment materials and methods were developed to enhance the abundance of glycopeptides in mass spectrometry analyses, such as lectin affinity chromatography [9], [10], [11], boronic acid affinity chromatography [12], [13], [14], [15], [16], hydrazide chemistry [17], [18] and hydrophilic interaction chromatography (HILIC) [19], [20], [21], [22], [23]. Among these methods, HILIC has gained increasing popularity because it shows high glycopeptide coverage, excellent reproducibility, high selectivity and mild enrichment conditions. However, the relatively low density of hydrophilic groups and limited surface area of the materials affected the binding capacity for glycopeptides. Therefore, it is imperative to develop novel HILIC materials with an improved functional group capacity, hydrophilicity, and strong retention of glycopeptides. Recently, zwitterionic molecule functionalized materials have revealed numerous advantages for glycopeptide enrichment due to the binding force between zwitterionic groups and hydrogen via electrostatically induced hydration, which is stronger than that for hydrogen-binding-induced hydration materials. Chen et al. developed zwitterionic magnetic nanoparticles by reflux-precipitation polymerization, and this new material exhibited high selectivity, extremely high detection sensitivity and large binding capacity for glycopeptide enrichment [24]. Jiang et al. prepared a zwitterionic polymer-coated graphene-oxide by employing cysteine as the zwitter-ionic polymer [25], and it exhibited good performance in glycopeptide enrichment from complex samples. As a familiar amino acid in organisms, cysteine possesses superhydrophilic properties, low cost and exists widely in nature. Therefore, the synthesis of zwitterionic polymers by cysteines would be attractive due to its potential as a convenient and effective glycopeptide enrichment method.

Over the past several decades, graphene oxide (GO) has become of increasing interest due to its unique chemical and physical properties [26], [27], [28], [29]. Apart from high surface area, excellent biocompatibility and good stability, GO is a novel one-atom-thick carbon nanosheet with hydroxyl and epoxy groups on the flat surface and carboxylic acid groups at the edges [30]. This special structure offers numerous reaction sites for functional material synthesis. A modified GO sheet with excellent hydrophilicity has been proven to be a suitable substrate for glycoproteomics research [31], [32]. Various methods were developed to graft hydrophilic groups on the GO nanosheet, among which the most widely used method was based on Au nanostructures that exhibited high reaction activity between Au and thiol groups [33]. However, a limited Au nanoparticle loading amount means that highly efficient glycopeptide enrichment remains challenging. Recently, one-dimensional gold nanowires (AuNWs), especially ultrathin nanowires with the diameter of 2 nm, have gained immense interest due to their high aspect ratios and potential applications in sensors, electronics and catalysis [34], [35]. Feng et al. synthesized single crystalline ultrathin gold nanowires at room temperature, and the resulting gold nanowires demonstrated an interesting application in surface-enhanced Raman scattering (SERS) [36]. Annamalai et al. immobilized the ultrathin gold nanowires on graphene oxide to prevent the Au nanocatalysts from aggregating. Stable ultrathin gold nanowires supported on a reduced graphene oxide exhibited outstanding electro-catalytic activity for borohydride oxidation [37]. Although a growing number of studies on gold nanowires have been made, there has been no report of the utilization of this novel nanowire in materials for proteomics research. The large surface area of ultrathin gold nanowires provides numerous reaction sites for the linkage of hydrophilic groups and might have great potential to improve the selectivity, sensitivity and binding capacity for glycopeptide enrichment.

Herein, we report for the first time a facile method for the synthesis of zwitterionic groups linked to magnetic graphene oxide (GO-Fe3O4/SiO2/AuNWs/L-Cys) via immobilized ultrathin gold nanowires. First, Fe3O4 was loaded onto the GO to form a high surface area magnetic platform for the subsequent synthesis. The subsequent magnetism made the separation of GO more convenient and efficient. Then, the GO-Fe3O4 was covered with a silica shell. This GO-Fe3O4/SiO2 not only showed less non-specific adsorption but also provided many active thiol groups so that the ultrathin gold nanowires could be stably immobilized. Ultrathin gold nanowires were immobilized via the linkage between the GO-Fe3O4/SiO2 and the hydrophilic components. Finally, L-Cys were loaded through an Au-S bond that endows the composite with excellent hydrophilic properties and a large binding capacity for glycopeptides. Several biological samples have been employed to evaluate the properties of GO-Fe3O4/SiO2/AuNWs/L-Cys composites for use in glycopeptide enrichment. The large concentration of hydrophilic groups enables this new HILIC material to achieve high efficiency, excellent selectivity, and a large capacity for glycopeptide enrichment from complex biological samples.

Section snippets

Materials and reagents

Iron(III) chloride hexahydrate (FeCl3·6H2O), sodium acetate (NaAc), (3-Mercaptopropyl)trimethoxysilane (MPTES), human immunoglobulin G (IgG), horse radish peroxidase (HRP), bovine serum album(BSA), Tetraaethylorthosilicate (TEOS), chloroauric acid (HAuCl4·3H2O), oleylamine (OA), triisopropylsilane (TIPS), 2,5-dihydroxybenzoic acid (DHB), ammonium solution, ammonium bicarbonate(NH4HCO3), triuoroacetic acid (TFA), formic acid (FA), l-Cysteine, TPCK-treated trypsin and peptide-N-glycosidase

Preparation and characterization of a GO-Fe3O4/SiO2/AuNWs/L-Cys composite

The process for the fabrication of GO-Fe3O4/SiO2/AuNWs/L-Cys composites is illustrated in Scheme 1. First, Fe3O4 nanoparticles were dotted onto GO via a solvothermal reaction to endow the composite with magnetism. Then, the GO-Fe3O4 particles were modified with silica shells via a sol-gel method. Subsequently, ultrathin Au nanowires were grafted onto the GO-Fe3O4/SiO2 composites through the specific interaction between SH groups and the Au nanowires. Finally, L-Cys was used as a hydrophilic

Conclusions

In summary, the ZIC-HILIC GO-Fe3O4/SiO2/AuNWs/L-Cys composites were successfully synthesized. The large surface area of the ultrathin Au nanowires increased the amount of zwitterionic molecules on the GO, resulting in excellent hydrophilicity. Glycopeptide enrichment experiments indicated that these composites could be applied to N-linked glycopeptide enrichment in both the tryptic digests of standard glycoproteins and complex biological samples. This work not only provides a novel material for

Conflict of interest

The authors declared no conflict of interest.

Acknowledgments

This work was supported by the National Key Program for Basic Research of China (Grants 2012CB910603, 2013CB911204, 2016YFA0501403 and 2016YFA0501300), the National Key Program for Scientific Instrument and Equipment Development (Grants 2012YQ12004407, 2011YQ06008408 and 2013YQ14040506), the National Natural Science Foundation of China (Grants 21275159 and 21235001).

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