Facile synthesis of thiol-polyethylene glycol functionalized magnetic titania nanomaterials for highly efficient enrichment of N-linked glycopeptides

Accepted Manuscript Title: Facile synthesis of thiol-polyethylene glycol functionalized magnetic titania nanomaterials for highly efficient enrichment of N-linked glycopeptides Authors: Jiawen Wang, Jizong Yao, Nianrong Sun, Chunhui Deng PII: DOI: Reference:

S0021-9673(17)31002-6 http://dx.doi.org/doi:10.1016/j.chroma.2017.07.020 CHROMA 358667

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

26-5-2017 5-7-2017 6-7-2017

Please cite this article as: Jiawen Wang, Jizong Yao, Nianrong Sun, Chunhui Deng, Facile synthesis of thiol-polyethylene glycol functionalized magnetic titania nanomaterials for highly efficient enrichment of N-linked glycopeptides, Journal of Chromatography Ahttp://dx.doi.org/10.1016/j.chroma.2017.07.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Facile synthesis of thiol-polyethylene glycol functionalized magnetic titania nanomaterials for highly efficient enrichment of N-linked glycopeptides Jiawen Wang1, Jizong Yao1, Nianrong Sun*1, Chunhui Deng2 1

Department of Chemistry, Fudan University, Shanghai 200433, China

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Department of Chemistry and Institutes of Biomedical Sciences, Collaborative Innovation

Center of Genetics and Development, Fudan University, Shanghai 200433, China. Corresponding author: Nianrong Sun, Email: [email protected] Chunhui Deng, Email: [email protected]; Fax: +86-21-65641740

Highlights 

A novel synthesis route for functionalization of thiol group containing compounds through interaction between titanium and sulfhydryl groups

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Simple manipulation under mild synthesis conditions

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Strong magnetic response for rapid separation from complex matrix

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Superior hydrophilicity for highly efficient enrichment of N-glycopeptides in both standard proteins and biological samples

Abstract: As protein N-glycosylation involved in generation and development of various cancers and diseases, it is vital to capture glycopeptides from complex biological samples for biomarker discovery. In this work, by taking advantages of the interaction between titania and thiol groups, thiol-polyethylene glycol functionalized magnetic titania nanomaterials (denoted as Fe3O4@TiO2@PEG) were firstly fabricated as an excellent hydrophilic adsorbent of N-linked glycopeptides. On one hand, the special interaction of titanium-thiol makes the synthetic manipulation simple and provides a new idea for design and synthesis of novel nanomaterials; on

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the other hand, strong magnetic response could realize rapid separation and the outstanding hydrophilicity of polyethylene glycol makes Fe3O4@TiO2@PEG nanomaterials show superior performance for glycopeptides enrichment with ultralow limit of detection (0.1 fmol/μL) and high selectivity (1:100). As a result, 24 and 33 glycopeptides enriched from HRP and IgG digests were identified respectively by MALDI-TOF MS, and 300 glycopeptides corresponding to 106 glycoproteins were recognized from merely 2 μL human serum, indicating a great potential of Fe3O4@TiO2@PEG nanomaterials for glycoproteomic research. Keywords: Enrichment, Functionalized magnetic titania nanomaterials, Glycopeptide, Hydrophilicity, MALDI-TOF MS

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1. Introduction Protein glycosylation, as one of the most prominent and prevalent protein post-translational modifications (PTMs) in eukaryotic cells, has aroused great interest among researchers for its vital roles in various cellular processes such as inter/intra-cellular signal transduction, cellular differentiations and so forth[1]. A great many researches, in the past decades, have revealed that a large number of cancers and immunodeficiency diseases generate accompanied with aberrant protein N-glycosylation or abnormal changes in number of N-linked glycoproteins expressed in body fluid, such as ovarian cancers and autoimmune rheumatic disease[2-4]. Hence, a comprehensive and high-throughput strategy for recognition of glycosylation site and the corresponding glycoforms is essential for the studies of the relationship between protein glycosylation and diagnosis or treatment of diseases. Until recently, mass spectrometry (MS) coupled separation strategies have been developed and applied to N-glycosylation profiling with the significant improvement of the sensitivity and accuracy of MS[5-7], though there are still various problems remaining to be solved, which involves the inherent low concentration of glycopeptides in complex biological samples and strong signal suppression of countless nonglycosylated peptides, let alone the poor ionization performance of glycopeptides. Therefore, for detailed study of relationship between glycopeptides and pathogenesis, it is indispensable to conduct an efficient and large-scale enrichment process before MS analysis. Up to now, varieties of approaches, such as hydrazine chemistry[8-10], boronic acid-based chemistry[11-13] and hydrophilic interaction liquid chromatography (HILIC)[14-16], have been immensely developed for glycopeptides enrichment. However, owing to the template-free biosynthesis of glycans, the high heterogeneity of protein N-glycosylation makes the complete enrichment still challenging[17]. Among these popular strategies, HILIC-based strategies stand

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out due to its multiple advantages of high enrichment efficacy, good reproducibility, simple operation, no bias towards different kinds of glycans, and more importantly, the high compatibility with post MS analysis. By now, various types of HILIC materials, including glucose[18], hydrophilic polymers[19, 20], metal–organic frameworks[21-23] and amino acid[24, 25], have been developed for glycopeptides enrichment. Despite of such great progress, many of them suffer from harsh reaction condition, cumbersome synthetic process or weak hydrophilicity, which hinder their application in the aspect of glycopeptides enrichment. For instance, polydopamine (PDA), as reducing and stabilizing agent for generation of Au particles, was reported to graft on magnetic graphene, which were subsequently used for attachment to thiol-containing L-cysteine (magG/PDA/Au/L-Cys) for selective enrichment of glycopeptides. The whole synthetic operation was tedious and time-consuming, leading to the relatively undistinguished enrichment efficiency of magG/PDA/Au/L-Cys[24]. Therefore, designing a simpler synthetic route to immobilize functionalization groups is great important for improving the enrichment efficacy of glycopeptides. Magnetic nanomaterials have been widely developed for biological applications, including enzyme immobilization[26, 27], drug delivery[28, 29] and cancer photothermal therapy[30, 31], since they can be simply and rapidly isolated from the sample solution based on their superparamagnetic behavior, thus saving times and leading to less loss in samples. In our preceding works, we have developed facile approaches for the synthesis of Fe3O4@TiO2 core-shell nanomaterials[32]. The well-defined titania shell protects the magnetic core from external environments, making it favorable substrate for further modifications. Polyethylene glycol (PEG), as a kind of polyether compound with flexible hydrophilic long chain, has wide applications from chemistry to manufacturing[7, 33, 34]. Commonly, it is modified by functional group like thiol to form thiol-PEG (SH-PEG), making for being immobilized on the

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surface of hydrophobic substrate or as a coupling agent for producing non-ionic surfactants[35, 36]. Besides, according to reports[37, 38], there are special interactions between titanium and the thiol group. Hence, in this work, for the first time, we successfully fabricated SH-PEG functionalized magnetic titania nanomaterials (denoted as Fe3O4@TiO2@PEG) by taking advantage of special interaction between titania and thiol compounds, which was a very facile functionalization route. With the rapid and effortless separation under external magnetic field, the enrichment process of Fe3O4@TiO2@PEG nanomaterials was extremely simplified. As the hydrophilic and biocompatible polymer, SH-PEG improves the binding capacity to glycopeptides and significantly reduce nonspecific adsorption of Fe3O4@TiO2@PEG, endowing it with outstanding hydrophilicity and high enrichment efficiency for glycopeptides from both of the standard sample such as tryptic HRP/IgG and complex bio-samples like human serum.

2. Experimental 2.1 Materials Titanium(IV) isopropoxide (TIPO), Ammonium bicarbonate (NH4HCO3), horseradish peroxidase (HRP), immunoglobulin G (IgG), myoglobin (Myo), dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic acid (TFA) and 2, 5-dihydroxy-benzoic acid (DHB) were purchased from SigmaAldrich (USA). Thiol-end polyethylene glycol (SH-PEG, Mn=5000) was purchased from Ponsure Biotechnology. Concentrated ammonia solution (28 wt%) was purchased from Tongsheng chemical reagent. PNGase F was purchased from Genetimes Technology. Acetonitrile (ACN) of chromatographically pure was purchased from Merck (Darmstadt, Germany). All deionized water in the experiment are acquired by Milli-Q system (Millipore, Bedford, MA). All of other chemicals

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are analytically pure. Human serum was provided by Shanghai Zhongshan Hospital from a healthy volunteer. 2.2 Apparatus The morphology of Fe3O4@TiO2@PEG nanomaterials was characterized by transmission electron microscopy (TEM) on a JEOL 2011 microscope (Japan) operating at 200 kV after the sample was homogeneously dispersed in ethanol by ultrasonication. Fourier-transform infrared (FT-IR) spectra performed on a Nicolet iS 10 Fourier spectrophotometer (USA) using KBr pellets to characterize the functional group on the surface of Fe3O4@TiO2@PEG nanomaterials. Powder X-ray diffraction (XRD) patterns were obtained by a Bruker D4 X-ray diffractometer with Nifiltered Cu Kα radiation (40 kV, 40 mA). X-ray photoelectron spectroscopy (XPS) spectra were adopted by ESCALab220i-XL electron spectrometer (VG Scientific) using 300 W Al Kα radiation. The base pressure was about 3×10-9 mbar. The binding energies were referenced to C 1s line at 284.8 eV from adventitious carbon. All MALDI-TOF MS experiments were carried out on AB Sciex 5800 MALDI TOF/TOFTM mass spectrometer (AB Sciex, USA) in a reflector positive mode with a 355nm Nd-YAG laser, 200 Hz frequency, and acceleration voltage of 20 kV. 2.3 Synthesis of Fe3O4@TiO2@PEG nanomaterials The designed synthetic protocol of Fe3O4@TiO2@PEG nanomaterials was displayed in Fig. 1. Fe3O4 magnetic nanoparticles of 200 nm diameter were prepared by the previous method[39]. Typically, 50 mg as-prepared Fe3O4 nanoparticles were dispersed in 475 mL ethanol containing 1.5 mL ammonia solution (28 wt%) with the help of ultrasonication for 10 min. Next, the mixture was mechanically stirred for 10 min before 5 mL ethanol solution containing 0.75 mL TIPO was added in drops. Then, the dispersion was heated to 45 °C under stirring for 24 h. The obtained particles were consequently washed with ethanol for several times, dried in vacuum at 50 °C

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overnight and calcined at 400 °C for 2 h to improve crystallinity (denoted as Fe3O4@TiO2). Finally, 10 mg Fe3O4@TiO2 nanoparticles were dispersed in 10 mL ethanol containing SH-PEG (10 mg) by ultrasonication at room temperature and continuously blended for 12 h. The final product (denoted as Fe3O4@TiO2@PEG) were washed with ethanol for three times and dried in vacuum at 50 °C overnight for the following experiments. 2.4 Preparation of protein digests and human serum sample Standard protein HRP, IgG or Myo was dissolved in 500 μL 50 mM NH4HCO3 (pH=8.3) and denatured in 95 °C for 8 min. After cooling down to room temperature, 500 μL Milli Q was added to make the final protein concentration 2 mg/mL, and the solution was ultimately incubated at 37 °C for 16h after trypsin was added at the protein/trypsin ratio of 40/1 (w/w). For preparation of human serum sample, 2 μL healthy human serum was mixed homogeneously with 16 μL 25 mM NH4HCO3 (pH=7.9) and the mixture was centrifuged at 12,000 rpm for 2 min. Afterwards, the obtained supernatant was reduced by 10 mM DTT at 60 °C for 30 min and cysteines were alkylated at 37 °C by 20 mM IAA for 1 h in the dark. The solution was then diluted five folds with 25 mM NH4HCO3 and trypsin (protein/Trypsin=40/1, w/w) was added to proceed protein digestion at 37 °C for 16 h. Finally, the digested samples were lyophilized and stored in 20 °C for further use. 2.5 Protocol of hydrophilic enrichment 100 μg Fe3O4@TiO2@PEG nanomaterials were added into 50 μL loading buffer (ACN/H2O/TFA=80/19.9/0.1, v/v/v, containing 10 mM NH4HCO3) with 100 fmol/ μL HRP (or IgG) digests. After incubated at 37 °C for 15 min, the nanoparticles were separated from supernatant by additional magnetic field and washed with 200 μL washing buffer (ACN/H2O/H3PO4=80/19.5/0.5, v/v/v) for three times. Afterwards, the obtained glycopeptides

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were eluted with 8 μL ACN/H2O/TFA (50/49.5/0.1, v/v/v, containing 10 mM NH4HCO3) at 37 °C for 20 min. At last, 1 μL eluent and 1 μL matrix solution was dropped and dried for MALDI-TOF MS analysis. For hydrophilic enrichment of glycopeptides from serum sample, the lyophilized serum digestion was redissolved in 100 μL loading buffer which contains 500 μg Fe3O4@TiO2@PEG nanomaterials. The dispersion was incubated at 37 °C for 30 min and rapidly separated from solution by magnet followed by washing step for several times. 30 μL eluting buffer was used twice to elute the glycopeptides as many as possible. The eluent was lyophilized and deglycosylated by PNGase F for Nano-LC-MS/MS analysis.

3 Results and discussion 3.1 Characterization of Fe3O4@TiO2@PEG nanomaterials The morphology of Fe3O4@TiO2@PEG nanomaterials was firstly characterized by transmission electron microscopy (TEM). It is difficult to determine their boundries from the TEM images since both Fe3O4 and TiO2 are metal oxide which means their image contrasts are similar. While from Fig. S1A(ESI†), we could observe that the diameter of Fe3O4 nanoparticles was about 200 nm. After condensation of TiO2, the diameter of nanoparticles increased to about 350 nm (Fig. S1B, ESI†). Judging from the above points, we could conclude that the Fe3O4 nanoparticles were wellencapsulated in titania shell, and the thickness of TiO2 was around 75 nm. After modified by SHPEG, the Fe3O4@TiO2@PEG nanomaterials still kept the spherical morphology and it could be clearly observed that the boundaries of Fe3O4@TiO2@PEG nanomaterials became blurred (Fig. 2), inferring that PEG was successfully modified. This could be further confirmed by Fourier Transform Infrared (FTIR) spectroscopy. As seen in Fig.3, the functional groups on Fe3O4@TiO2@PEG nanomaterials were further characterized by FTIR, and the precursor

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Fe3O4@TiO2 nanoparticles were also characterized for comparison. From Fig. 3A, the broad band between 500 cm-1 and 700 cm-1 in both curves was attributed to the Ti-O-Ti stretching modes according to the literature[40]. After functionalized by SH-PEG, several new vibrational absorption peaks emerged that peak at 2882 cm-1 was attributed to the C-H stretching vibration, peak at 1468 cm-1 was attributed to -CH2 bending vibration adsorption and a strong peak at 1106 cm-1 was due to the C-O-C asymmetric vibration adsorption, indicating the successfully modification of thiol-PEG on Fe3O4@TiO2 nanomaterials[36].

Moreover, EDX analysis of the Fe3O4@TiO2@PEG nanomaterials revealed the existence of Fe, Ti, Cu, C, O and S (The strong Cu peaks were from the copper grid for TEM), which confirmed the successful synthesis of Fe3O4@TiO2@PEG nanomaterials indirectly (Fig. S4, ESI†). Additionally, X-ray photoelectron spectroscopy (XPS) was also applied to characterize the surface elements of Fe3O4@TiO2@PEG nanomaterials. Compared with the spectrum of Fe3O4@TiO2 (Fig. 3C), the peaks of C 1s, O 1s, S 2p3, Fe 2p, Ti 2p3 and Ti 3p could be evidently observed in Fig. 3D. After immobilization of SH-PEG, the C 1s/O 1s value was altered from 2.33 to 3.12, demonstrating the successful bonding of SH-PEG. As shown in Fig. 3B, the Ti 2p3/2-1/2 XPS peak could be fitted into two doublets assigned to Ti-O bond and Ti-S bond, respectively, which directly confirmed the bonding mode between titania and SH-PEG.[41, 44](binding energy could be found in Table S1, ESI†). Afterwards, Thermogravimetric Analysis (TGA) was employed to evaluate the thermal stability of Fe3O4@TiO2@PEG nanocomposites and the loading amount of SH-PEG chains. As seen in Fig. S5 (ESI†), there was no obvious weight loss in the curve of Fe3O4@TiO2 nanocomposites, indicating the outstanding thermal stability of them. While for the red curve, an

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evident mass loss could be observed after 300 °C and the content of SH-PEG in Fe3O4@TiO2@PEG nanocomposites was calculated as about 6.3 wt% when taking consideration of the data at 800 °C. Meanwhile, wide-angle X-ray Diffraction (XRD) pattern of Fe3O4@TiO2 nanomaterials was displayed in Fig. S2 (ESI†), peaks in which are well-matched to magnetite and anatase in previous literatures[40], and as shown in Fig. S3 (ESI†), Fe3O4@TiO2@PEG nanomaterials could be well dispersed in water and rapidly separated from water under external applied magnetic field, the magnetic property of which was demonstrated by SQUID magnetometer. The saturation magnetization value of Fe3O4@TiO2@PEG nanomaterials was 44.1 emu·g-1. 3.2 Optimization of incubation conditions of Fe 3O4@TiO2@PEG nanomaterials. The efficient procedure for the enrichment of N-glycopeptides by utilizing Fe3O4@TiO2@PEG nanomaterials was displayed in Fig. 4. For HILIC materials, it has been widely reported that it is vital to slightly adjust the concentration of acetonitrile (ACN) to give a quite appropriate hydrophobic environment for them to capture glycosylated peptides[42]. Hence, above all, the incubation condition of Fe3O4@TiO2@PEG nanomaterials was evaluated by capturing glycopeptides from 100 fmol/μL horseradish peroxidase (HRP) digest in a series of loading buffer. ACN/H2O/TFA (75/24.9/0.1, v/v/v, containing 10 mM NH4HCO3), ACN/H2O/TFA (80/19.9/0.1, v/v/v, containing 10 mM NH4HCO3) and ACN/H2O/TFA (85/14.9/0.1, v/v/v, containing 10 mM NH4HCO3) as loading buffer candidates were applied. As is shown in Fig. S6 (ESI†), there were apparent lower signal intensities of glycopeptides observed from 75% ACN/ 0.1% TFA (containing 10 mM NH4HCO3). Both of the number and peak intensity of glycopeptides improved significantly with the increasing concentration of acetonitrile, while there were more non-glycosylated peptides were adsorbed when using 85% ACN/ 0.1% TFA (containing 10 mM NH4HCO3) as loading buffer,

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thus ACN/H2O/TFA (80/19.9/0.1, v/v/v) with 10 mM NH4HCO3 was applied as loading buffer for the following enrichment of glycopeptides. Similarly, in another investigation, ACN/H2O/TFA (50/49.9/0.1, v/v/v) with 10 mM NH4HCO3 was chosen as the eluting buffer since the interference of non-glycosylated peptides is minimal. Moreover, we found that 15 min incubation is far enough for Fe3O4@TiO2@PEG to enrich glycopeptides since the number and signal intensity of glycopeptides were almost equally to those incubated for 60 min, which demonstrated the rapid enrichment ability of Fe3O4@TiO2@PEG (Fig. S7, ESI†).

3.3 N-glycopeptides enrichment from standard proteins by Fe3O4@TiO2@PEG nanomaterials. After optimization of incubation condition, Fe3O4@TiO2@PEG nanomaterials were applied to adsorption of glycopeptides from 100 fmol/μL HRP digestion. As shown in Fig. 5A, only 5 glycopeptides with low intensity were detected by direct MS analysis and there were a great many non-glycopeptides in the MS spectra. While applying Fe3O4@TiO2@PEG nanomaterials to the same concentration HRP digest, 24 glycopeptides with enhanced signal-to-noise ratio were detected by MS and most of non-glycosylated peptides were removed (Fig. 5C, detailed information was listed at Table S2, ESI†). For comparison, Fe3O4@TiO2 nanocomposites were also applied to the same process (Fig. 5B), and its poor adsorption ability for glycopeptides demonstrated a good hydrophilic property of Fe3O4@TiO2@PEG nanomaterials. Even if the concentration of HRP digest decreased to 0.1 fmol/μL, 6 glycopeptides could still be identified, which means Fe3O4@TiO2@PEG had a super low limit of detection and could be qualified as a brand-new platform for glycopeptides analysis (Fig. S8, ESI†). Moreover, the batch-to-batch reproducibility of Fe3O4@TiO2@PEG nanomaterials was tested. As seen in Fig. S9 (ESI†) after

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enrichment by different batches of Fe3O4@TiO2@PEG, a similar number of N-glycopeptides with fairly stable relative intensities were identified, indicating the controllability of preparation of Fe3O4@TiO2@PEG nanomaterials based on this protocol. Besides, the reproducibility of the same batch of Fe3O4@TiO2@PEG nanomaterials was also tested. And the results suggested Fe3O4@TiO2@PEG nanomaterials could be reused for N-glycopeptides enrichment. (Fig. S10, ESI†) To demonstrate that Fe3O4@TiO2@PEG nanomaterials have no bias towards different kinds of glycans, tryptic IgG (100 fmol/μL) which contains different glycoform from HRP was employed. Before enrichment, merely 3 glycopeptides were detected with low peak intensities and a great amount of interference was seen in Fig. 6A while 33 high signal-to-noise glycopeptides were obtained after enriched by Fe3O4@TiO2@PEG nanomaterials (Fig. 6B, detailed information is listed in Table S3, ESI†). Furthermore, peptides mixture of Myo (typical non-glycosylated protein) and HRP with different molar ratio were employed to test the enrichment selectivity for Fe3O4@TiO2@PEG nanomaterials. As shown in Fig. 7, before enriched by Fe3O4@TiO2@PEG, lots of Myo peptides were observed whereas few glycopeptides were found because of the suppression of Myo peptides when HRP and Myo peptides at the ratio of 1 to 10. But after enrichment by Fe3O4@TiO2@PEG (Fig. 7C), evident peaks of 23 glycopeptides were detected by MS. When the molar ratio further increased to 1:100, 23 glycopeptides could still be clearly observed after enrichment by Fe3O4@TiO2@PEG (Fig. 7D), which indicated the great potential of Fe3O4@TiO2@PEG nanomaterials for glycopeptides enrichment from complex biological samples. 3.4 N-glycopeptides enrichment from human serum by Fe3O4@TiO2@PEG nanomaterials.

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Encouraged by their excellent selectivity and low limit of detection of Fe3O4@TiO2@PEG nanomaterials, we further applied them to enrich glycosylated peptides from biological samples. Human serum, as typical specimen in clinic diagnosis, is widely used for prognosis determination and symptom profiling. It has aroused wide interests for biomarkers discovery since some specific proteins, especially glycosylated proteins, would be leaked from diseased organs into the blood stream[43, 44]. In this work, Fe3O4@TiO2@PEG nanomaterials were employed in glycoproteomics analysis of healthy human serum. After enriched by Fe3O4@TiO2@PEG, Nlinked glycans were removed from the obtained glycopeptides for nano-LC/MS/MS analysis. A total of 300 unique glycopeptides corresponding to 106 glycoproteins enriched from only 2 μL human serum was finally identified, far more than previously reported[19, 45, 46].(detailed information is listed in Table S4&S5, ESI†) All these results mentioned above suggested that the Fe3O4@TiO2@PEG nanomaterials exhibited superior performance for enriching glycopeptides from complicated biological samples, and could be expected for further analysis of aberrant glycopeptides for discovery of disease biomarkers.

4. Conclusion In conclusion, the Fe3O4@TiO2@PEG nanomaterials with excellent hydrophilicity were firstly synthesized for glycopeptides enrichment from complex biological sample. The facile and fast combination between titanium and thiol simplified the synthetic process, which not only provided new train of thoughts for design and synthesis of nanomaterials with various purposes, but also allowed great amount of PEG modified on the surface of the Fe3O4@TiO2 that making for the advanced hydrophilicity of Fe3O4@TiO2@PEG nanomaterials. With a variety of merits such as advanced hydrophilicity, easy-to-prepare and strong magnetic response, the Fe3O4@TiO2@PEG nanomaterials were verified distinguished enrichment performance for not only the standard

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samples but also the real biological samples like human serum. 300 glycopeptides within 106 glycoproteins were detected from merely 2 μL human serum. The superior enrichment ability makes it a prospective candidate for glycosylation analysis based on MS strategies, implying its great potential in early diagnosis of diseases and cancers. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21425518, 21405022 and 21675034) and National Basic Research Priorities Program of China (2013CB911201). Appendix A. Supplementary data 
 Supplementary data associated with this article can be found, in the online version, at XXX

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Fig. 1 Schematic diagram of the preparation of Fe3O4@TiO2@PEG nanomaterials

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Fig. 2 The TEM images of Fe3O4@TiO2@PEG nanomaterials.

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Fig. 3 (A) The FTIR spectra of Fe3O4@TiO2 and Fe3O4@TiO2@PEG nanomaterials. The XPS spectra of (B) Ti 2p3; (C) Fe3O4@TiO2 nanocomposites; (D) Fe3O4@TiO2@PEG nanomaterials.

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Fig. 4 The specific enrichment workflow for N-glycopeptides by Fe3O4@TiO2@PEG nanomaterials.

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Fig. 5 MALDI-TOF MS spectra of 100 fmol/μL HRP digest (A) before enrichment (B) enriched by Fe3O4@TiO2 nanomaterials (C) enriched by Fe3O4@TiO2@PEG nanomaterials. Glycopeptides are marked with

.

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Fig. 6 MALDI-TOF MS spectra of N-linked glycopeptides enriched from 100 fmol/μL IgG digests: (A) before enrichment; (B) after enriched by Fe3O4@TiO2@PEG nanocomposites. Glycopeptides are marked with

.

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Fig. 7 MALDI-TOF MS spectra of mixture of tryptic HRP and Myo before enrichment: (A) 1:10; (B) 1:100. MALDI-TOF MS spectra of the identified glycopeptides enriched by Fe3O4@TiO2@PEG from the tryptic mixture of HRP and Myo with the molar ratio of (C) 1:10; (D) 1:100. Glycopeptides are marked with

.

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