Two-Dimensional MoS2-Based Zwitterionic Hydrophilic Interaction

May 9, 2018 - ... College of Chemistry and Materials Science, Northwest University ... State Key Laboratory of Proteomics, National Center for Protein...
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2D-MoS2-Based Zwitterionic (ZIC)-HILIC Material for the Specific Enrichment of Glycopeptides Chaoshuang Xia, Fenglong Jiao, Fangyuan Gao, Heping Wang, Yayao Lv, Yehua Shen, Yangjun Zhang, and Xiaohong Qian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00461 • Publication Date (Web): 09 May 2018 Downloaded from http://pubs.acs.org on May 10, 2018

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Analytical Chemistry

2D-MoS2-Based Zwitterionic (ZIC)-HILIC Material for the Specific Enrichment of Glycopeptides *

Chaoshuang Xiaa,b, Fenglong Jiaob, Fangyuan Gaob, Heping Wangb,c, Yayao Lvb, Yehua Shen a, Yangjun Zhang*b, Xiaohong Qianb a

Key Laboratory of Synthetic and Natural Function Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi'an 710069, China. b

State Key Laboratory of Proteomics, National Center for Protein Science Beijing, Beijing Institute of Radiation Medicine, Beijing 102206, China.

c

School of Chemistry & Chemical Engineering, Ankang University, Ankang, Shaanxi, 725000, China

KEYWORDS: 2D nanosheet, Molybdenum disulfide (MoS2), glycopeptides, enrichment, Exosome ABSTRACT: Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS2-Au-NP-Lcysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS2 nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to a commercial ZIC-HILIC materials, the novel nanomaterials exhibited excellent enrichment performance with ultra-high selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g-1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptides from 775 glycoproteins were identified in three replicate analyses of 50 µg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics.

including the inherent low abundance and high dynamic range of glycosylated proteins in complex biological samples, the 9,10 and the frelow ionization efficiency of glycopeptides, quent strong suppression of the MS signals of glycopeptides in the presence of non-glycopeptides and salts. Therefore, it is of critical importance to develop a method for the effective enrichment of glycopeptides from a complex biological mixture prior to MS analysis. To address this problem, numerous studies have been devoted to developing strategies to enrich glycopeptides prior to 11 MS detection, such as lectin affinity chromatography, hydrophilic interaction liquid chromatography (HILIC), boronic 12 acid affinity and hydrazine chemistry. Among these methods, lectins have been widely used to isolate, purify and characterize glycoproteins and glycopeptides, but theirs narrow substrate binding affinities for various glycan still need to be 2 improved. Boronate-functionalized materials have exhibited high potential in glycoproteome analysis since it is wellknown that the cis diols of glycans can form reversible covalent diester bonds with boronic acid groups. However, this method is limited by low selectivity and sensitivity in complex

INTRODUCTION Of the more than 300 known protein modifications, glycosylation is one of the most abundant and complex post1,2 translational modifications (PTMs). The glycosylation of proteins can endow the precursor protein with new properties including cellular trafficking and localization, binding specificity and thermodynamic stability. As a result, glycoproteins on the living cell surface play crucial biological and physiological roles, such as cell adhesion, molecular recognition, 3,4 signal transduction and immune response. Meanwhile, aberrant glycosylation has been found to be a hallmark of many diseases, including neurodegenerative diseases, immune defi5,6 ciencies, and many cancers. Therefore, glycoproteins have great potential as disease biomarkers from a pharmacological point of view. Consequently, substantial effort has been directed towards the accurate characterization of a cell’s glycoproteome. In recent decades, mass spectrometry (MS) has become an effective tool for the in-depth profiling of glycoproteomes for 7,8 biomarker research. Unfortunately, there are challenges,

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conditions. In addition, hydrazide chemistry is used extensively to isolate, identify, and quantify glycopeptides effectively; however, this method has some limitations, including timeconsuming and lengthy chemical procedures, low selectivity and sensitivity, lack of specificity for glycopeptide enrichment, and side reactions between glycan moieties and sodium 2 13 periodate. Another enrichment strategy based on HILIC, isolating glycopeptides and non-glycopeptides by their hydrophilicity differences, has become a popular method for glycopeptide enrichment. Moreover, HILIC can enrich different glycopeptides in an unbiased manner without damaging the structure of the glycans of glycopeptides. Therefore, several 14,15 16 materials, such as silica, sepharose, magnetic nanopar17 18 ticles, graphene oxide (GO) and metal-organic frame19-21 works, have been modified by hydrophilic groups for glycopeptide enrichment by the HILIC method. However, many factors, including relatively low specific surface area and large steric hindrance of these materials, frequently cause these methods to suffer from low enrichment selectivity and poor performance under harsh conditions. Therefore, there is great demand to develop a material with excellent performance for glycopeptide enrichment. Molybdenum disulfide (MoS2), a typical graphene-like twodimensional (2D) layer material, has attracted great scientific 22-25 and technological interest due to its unique structural properties and other outstanding properties, including high surface area, biocompatibility and innocuity and outstanding thermal, mechanical and solvent stability. As a result, MoS2 has received increasing attention in different areas such as 26 27 electrochemical energy storage, hydrogen evolution, lu28 29 minescent sensing, photovoltaic applications, 30,31 23,29,32,33 biosensing and disease theranostics. Although there are many studies on MoS2 nanocomposites, the utilization of this novel material for the preparation of zwitterionic (ZIC)-HILIC composites has not been reported. Considering the above qualities, MoS2-Au-NP-L-cysteine nanocomposites were synthesized for the first time. Briefly, gold nanoparticles (Au-NPs) were self-assembled on the edge sites and defective sites of MoS2 nanosheets via a simple and robust ‘Au-S’ bond, and a large amount of L-cysteine was immobilized through a further ‘Au-S’ bond because Lcysteine possesses excellent hydrophilicity and high selectivity 34,35 for glycopeptide enrichment. The two-dimensional ultrathin planar structure not only provides a large specific surface area but also eliminates steric hindrance between the material and the glycopeptides. Moreover, it is easy to prepare the composite by a clever synthesis procedure based on two ‘AuS’ reactions. Thus, the greatest advantage of the obtained nanomaterials is the incorporation of the merits of the individual components. The synthesized MoS2-Au-NP-L-cysteine nanocomposites exhibited outstanding hydrophilicity, good biocompatibility and innocuity. As a consequence, these features lead to their wonderful glycopeptide enrichment ability in MSbased glycoproteomics studies on real biological samples.

nano-LC-ESI-MS/MS analysis and database searching are described in Supporting Information. Tryptic digest of model glycoproteins and proteins of HeLa cell exosomes. One milligram of human serum immunoglobulin G (IgG) or horseradish peroxidase (HRP) was dissolved in 1 mL of NH4HCO3 buffer solution (50 mM, pH=8.0), denatured at 100 °C for 5 min and then incubated with 20 µg of trypsin for digestion at 37 °C overnight. Next, the tryptic digests were desalted by a Sep-Pak C18 SPE column (Waters) and stored at -20 °C until further use. Exosomes are small membrane vesicles (30-150 nm) secreted by various types of normal and disease-related cells. They contain functional biomolecules (including proteins, lipids, RNA and DNA) that can be horizontally transferred to recipient cells, which are widely researched as a potential source of biomarkers for bioanalytical applications and disease theranostics.36-39 For complex samples, the protein mixture from HeLa cell exosomes was extracted by following a literature procedure.36,37 The protein mixture (500 µg) was dissolved in urea solution (8 M), lysed in an ice bath for 30 min, and reduced by DTT (10 mM) at 37 °C for 4 h, then alkylated by IAA (50 mM) in the dark at room temperature for 1 h. Then, the solution was mixed with 10 µg of trypsin for digestion at 37 °C overnight. Next, the tryptic digests were desalted by Sep-Pak C18 SPE column and stored at -20 °C until further use. Protocol of enrichment process. Twenty micrograms of MoS2/Au-NP-L-cysteine nanomaterials was suspended in 100 µL of loading buffer (ACN/H2O/TFA=86/13.9/0.1, v/v/v) containing 1 µg of IgG tryptic digest or 0.5 µg of HRP tryptic digest. The mixture was gently incubated on a platform shaker for 30 min at room temperature. Next, the nanomaterials were separated from the mixture by centrifugation at 1,500 ×g for 2 min, followed by washing three times with 100 µL of washing buffer (ACN/H2O/TFA=86/13.9/0.1, v/v/v). Then, the captured glycopeptides were eluted twice with 20 µL of eluting buffer (ACN/H2O/TFA=30/69/1, v/v/v) for 10 min at room temperature. Finally, the eluent was analysed by MALDI-TOF MS or deglycosylated for nano-LC-ESI-MS/MS analysis. For glycopeptide enrichment from the proteins of HeLa cell exosomes, 50 µg of the abovementioned lyophilized tryptic digest was redissolved in 200 µL of loading buffer (ACN/H2O/TFA=86/13.9/0.1, v/v/v), and then 1 mg of MoS2/Au-NP-L-cysteine nanomaterials was added to the solution. The mixture was incubated on a platform shaker for 1 h at room temperature. Then, the nanomaterials were isolated from the mixture by centrifugation at 1,500 ×g for 5 min and washed three times with 200 µL of washing buffer (ACN/H2O/TFA=86/13.9/0.1, v/v/v). Then, the captured glycopeptides were eluted with 3×100 µL of eluting buffer (H2O/FA=95/5, v/v) for 10 min at room temperature. Finally, the three fractions of eluted glycopeptides were mixed, lyophilized and deglycosylated for nano-LC-ESI-MS/MS analysis. RESULTS AND DISCUSSION Characterization of MoS2, MoS2/Au-NP nanocomposites and MoS2/Au-NP-L-cysteine nanocomposites. The synthesis procedure of MoS2/Au-NP-L-cysteine nanocomposites is displayed in Scheme 1a. The morphology of the MoS2 nanosheets was characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The SEM (Fig. 1a)

EXPERIMENTAL SECTION The subsection of materials and chemicals, characteraztion of materials, preparation of MoS2/Au-NP-L-cysteine nanocomposites, deglycosylation of N-link glycopeptides, binding capacity, enrichment recovery, MALDI-TOF MS analysis,

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Analytical Chemistry MoS2/Au-NP nanocomposites shows an extra peak at 521 cm, which is due to the Au-S bond stretching. For the final composites, numerous peaks appeared from 500 cm-1 to 1500 cm-1 in both the L-cysteine and the MoS2/Au-NP-L-cysteine spectra, which suggested that L-cysteine was successfully grafted onto the MoS2/Au-NP nanocomposites.

and TEM (Fig. 1d) images showed that the MoS2 nanosheets were a typical layered nanostructure with transparent silk waves, and the two-dimensional MoS2 nanosheets had a size range of 70-300 nm. After decoration with Au-NPs, Fig. 1b and Fig. 1e show that Au-NPs with an average diameter of 10 nm were dispersed homogeneously on the surface of the MoS2 nanosheets, indicating that the MoS2/Au-NP nanocomposite was successfully synthesized. Finally, the nanocomposite was thicker (Fig. 1c) and its surface became rough (Fig. 1f) than MoS2/Au-NP, indicating an amount of L-cysteine was anchored on the MoS2/Au-NP surface successfully.

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Scheme 1. (a) the synthesis procedure of MoS2/Au-NP-Lcysteine, (b) the enrichment procedure for glycopeptides.

Figure 2. Characterization of MoS2, MoS2/Au-NP and MoS2/Au-NP-L-cysteine. (a) FT-IR spectra of MoS2, MoS2/Au-NP, L-cysteine and MoS2/Au-NP-L-cysteine, respectively. (b) X-ray diffraction patterns of MoS2 and MoS2/Au-NP, respectively. (c) TAG curves of MoS2, MoS2/Au-NP and MoS2/Au-NP-L-cysteine, respectively. Fig. 2b shows the X-ray diffraction (XRD) patterns of the freshly prepared MoS2 nanosheets and MoS2/Au-NP nanocomposites. For the freshly prepared MoS2 nanosheets, sharp peaks at 14° (marked by 002), 33° (101), 40° (222) and 59° (110) are observed. The diffraction profiles demonstrated the phase purity of MoS2 with a crystallized hexagonal structure (JCPDS 37-1492). After loading with Au-NPs, the XRD pattern of the MoS2/Au-NP nanocomposites exhibited new conspicuous peaks at 2θ values of 38.3°, 44.6°, 64.5°and 77.6°, which are assigned to the (111), (200), (220) and (311) of crystalline Au (JCPDS 01-1172), respectively. The characteristic peaks indicated that Au-NPs were decorated on the original MoS2 nanosheets. To better demonstrate the combination, thermogravimetric analysis (TGA) was carried out from 25 °C

Figure 1. SEM image and TEM image of the (a, d) MoS2, (b, e) MoS2/Au-NP and (c, f) MoS2/Au-NP-L-cysteine. According to FT-IR analysis (Fig. 2a), the peak at 468 cm−1 corresponds to Mo-S vibration. The FT-IR spectrum of

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to 800 °C under a N2 atmosphere at a heating rate of 10 °C min-1. As shown in Fig. 2c, the weight loss of the MoS2 nanosheets, MoS2/Au-NP nanocomposites and MoS2/Au-NPL-cysteine nanocomposites were 30%, 7% and 70%, respectively. The results indicated that the thermal stability of MoS2 nanosheets was enhanced after loading with Au-NPs. Furthermore, a slight mass loss before 220 °C in the curve of the MoS2/Au-NP-L-cysteine nanocomposites can be observed, and then a weight loss of 67% occurred. Thus, the content of L-cysteine in the MoS2/Au-NP-L-cysteine nanocomposites was calculated to be 67%. The above results indicated that the MoS2/Au-NP-L-cysteine nanocomposites have good thermal stability. In addition, a substantial amount of L-cysteine was grafted to the MoS2/Au-NP nanocomposites, which is attributed to the large surface area of MoS2. Enrichment performance of MoS2/Au-NP-L-cysteine nanocomposites for glycopeptides in model glycoproteins. The enrichment procedure for glycopeptides by MoS2/Au-NPL-cysteine nanocomposites is illustrated in Scheme 1b and includes three steps: loading, washing and eluting for detection by MS. In addition, glycopeptides could be retained on the surface of the nanocomposites, while non-glycopeptides could be washed away. The IgG tryptic digest was first employed as model glycopeptides to evaluate the enrichment performance of MoS2/Au-NP-L-cysteine nanocomposites. As shown in Fig. 3a, only 5 glycopeptides can be detected directly in the IgG tryptic digest without enrichment because the high abundance of non-glycopeptides inhibited the detection of glycopeptides. After enrichment by MoS2/Au-NP-L-cysteine nanocomposites, 32 glycopeptides can be detected because the non-glycopeptides were almost completely removed (Fig. 3b Table S1 and Table S2). For comparison, the commercial ZIC®-HILIC material was employed to capture glycopeptides from the IgG tryptic digest, and only 21 glycopeptides could be detected (Fig. 3c). Meanwhile, the above results were better than those previously reported with GO-Fe3O4/SiO2/AuNPs/LCys ZIC-HILIC (26),18 ZIC-HILIC polymer particles Poly(MBAAm-co-MAA)@L-Cys (17)35 and 40 Fe3O4@PGMA@Au-L-cys (28) for IgG tryptic digest enrichment. And the elution fraction of glycopeptide enrichment with MoS2/Au-NP-L-cysteine nanocomposites was characterized using nano-LC-ESI-MS/MS and analyzed with Byonic software. Before glycopeptide enrichment, 12 glycopeptides and 48 non-glycopeptides from IgG tryptic digests could be detected by nano-LC-ESI-MS/MS (Table S3), while 51 glycopeptides and 7 non-glycopeptides from IgG tryptic digests could be detected by nano-LC-ESI-MS/MS (Table S4) after glycopeptide enrichment. The results confirmed that the good performance of MoS2/Au-L-cysteine nanomaterials for selective enrichment of glycopeptides has been achieved. These results demonstrated that the MoS2/Au-NP-L-cysteine nanocomposites have high enrichment efficiency for glycopeptides from the IgG tryptic digest. For unambiguous verification, all glycopeptides captured after enrichment by MoS2/Au-NP-L-cysteine nanocomposites were deglycosylated by PNGase F. As shown in Fig. 3d, four deglycopeptides could be detected, which further confirmed that the peaks could be attributed to the glycopeptides. The results indicated that the MoS2/Au-NP-L-cysteine nanocomposites have excellent performance for glycopeptides. Next, the binding capacity of MoS2/Au-NP-L-cysteine nanocomposites was investigated. Different amounts of MoS2/Au-NP-L-cysteine nanocomposites were incubated with

a fixed amount of IgG tryptic digest (3 µg). After enrichment, the signal intensity of six selected glycopeptides reached a maximum when the amount of MoS2/Au-NP-L-cysteine nanocomposites was 25 µg (Fig. S1). In addition, the capacity for glycopeptides could be calculated to be 120 µg mg-1, owing to the high amount of grafted L-cysteine and its excellent hydrophilicity. Meanwhile, the enrichment recovery was also determined using the stable isotope dimethyl labelling technique according to the literature.41 As shown in Table S5, the average recovery obtained was 93.7%±3% by using MALDI-TOF MS analysis. Furthermore, in order to achieve more accurate quantitative results, the parallel reaction monitoring (PRM) mass spectrometry method was employed to quantify the recovery of glycopeptide enrichment. As shown in Table S6, the recovery of glycopeptide enrichment is 93.2%±7%. Both similar results indicated that MoS2/Au-L-cysteine nanomaterials have excellent recovery for the selective enrichment of glycopeptides. Meanwhile, the above results are superior to those previously reported with cysteine-functionalized MOFs (80%),21 CS@PGMA@IDA-Ti4+ (75.4%),42 Fe3O4@MPS@PMAC (82%),17 GO-PEI-Au-L-Cys (79.4%)43 and 40 Fe3O4@PGMA@Au-L-Cys (89.8%) for the glycopeptide enrichment of a IgG tryptic digest.

Figure 3. MALDI-TOF MS spectra of IgG tryptic digest (1 µg). (a) direct analysis, (b) after enrichment by MoS2/Au-NPL-cysteine, (c) after enrichment by ZIC®-HILIC, (d) after enrichment by MoS2/Au-NP-L-cysteine and deglycostlation by PNGase F. The detection sensitivity of glycopeptides enriched by MoS2/Au-NP-L-cysteine nanocomposites was also evaluated due to the ultra-low abundance of glycoproteins in complex bio-samples. As presented in Fig. 4a, 28 glycopeptides could be detected in 0.67 pmol of IgG tryptic digest. When the amount of IgG tryptic digest was 20 fmol, 5 glycopeptides

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Analytical Chemistry could be detected (Fig. 4b). Even when the amount of IgG tryptic digest was as low as 10 fmol, there was still one glycopeptide that could be detected with a high S/N ratio (Fig. 4c). For comparison, the commercial ZIC®-HILIC material was also used to enrich the glycopeptides under the same conditions. Unfortunately, there were only two glycopeptides that could be detected when the amount of IgG tryptic digest was 50 fmol (Fig. 4e), and no glycopeptide could be detected when the amount of IgG tryptic digest was 10 fmol (Fig. 4f). These results are superior to those previously reported with immobilized metal ion affinity chromatography (IMAC)- and hydrophilic interaction liquid chromatography (HILIC)-based functional materials (CS@PGMA@IDA-Ti4+, 45 fmol).42 Therefore, the nanocomposites showed high detection sensitivity for enriched glycopeptide, which was attributed to their good water dispersibility.

To evaluate the selectivity of MoS2/Au-NP-L-cysteine nanocomposites for the selective enrichment of glycopeptides, a mixture of IgG tryptic digest and BSA tryptic digest was incubated with the nanocomposites. As shown in Fig. 5, after enrichment by MoS2/Au-NP-L-cysteine nanocomposites, 24 glycopeptides can be detected when the mass ratio of IgG tryptic digest (3.3 pmol) to BSA tryptic digest was 1:1 (Fig. 5a). Furthermore, there were 7 glycopeptides that could be detected when the mass ratio were 1:100 (100 fmol IgG tryptic digest, Fig. 5b) and 1:200 (50 fmol IgG tryptic digest, Fig. 5c). Even when the ratio was as low as 1:500 (1:1250 molar equivalents of IgG tryptic digest to BSA tryptic digest, 20 fmol IgG digest), there were still 5 glycopeptides that could be observed at high intensities without any non-glycopeptide interference (Fig. 5d). For comparison, the commercial ZIC®-HILIC material was also used to capture glycopeptide under the same conditions. There were only 10 glycopeptides and 2 glycopeptides that could be detected when the mass ratio were 1:1 (3.3 pmol IgG tryptic digest, Fig. 5e) and 1:10 (500 fmol IgG tryptic digest, Fig. 5f), respectively. In addition, then, no glycopeptide could be observed when the mass ratio was 1:100 (100 fmol IgG tryptic digest, Fig. 5g). And the detailed information is provided in Table S2. The above results are better than those reported in previous publications with magnetic covalent organic frameworks (1:100),44 Fe3O4@mSiO2-IDA (1:200),15 cysteine-functionalized MOF (1:50),21 ZIC-HILIC polymer particles Poly(MBAAm-co-MAA)@L-Cys(1:10)35 and Fe3O4@PGMA@Au-L-cys (1:100).40 The interference experiment illustrated that MoS2/Au-NP-L-cysteine nanocomposites have extremely high detection sensitivity for glycopeptide enrichment. To further evaluate the universality of MoS2/Au-NP-Lcysteine nanocomposites for the hydrophilic enrichment of glycopeptides, both HRP tryptic digest and RNase B tryptic digest were also used as model sample for glycopeptide enrichment by MoS2/Au-NP-L-cysteine nanocomposites. The results showed that only 7 glycopeptides (Fig. 6a) and 2 glycopeptides (Fig. S2a) can be detected directly in the tryptic digest HRP and RNase B without enrichment, respectively, because the high abundance of non-glycopeptides inhibited the detection of glycopeptides. After enrichment by MoS2/Au-NPL-cysteine nanocomposites, 21 glycopeptides and 11 glycopeptides can be detected (Fig. 6b and Table S7, Fig. S2b and Table S8), respectively. Futhermore, the tryptic digests of 4 proteins (HRP, RNase B, IgG, BSA) were mixed at a mass ratio of 1:2:4:4 of HRP: RNase B: IgG: BSA, then 5 µg of the tryptic digest mixture was employed in glycopeptide enrichment. As shown in Fig. S3 and Table S9, only 2 glycopeptides can be detected directly by MALDI-TOF MS. After enriched by MoS2/Au-NP-L-cysteine nanocomposites, 36 glycopeptides could be detected by MALDI-TOF MS. For comparison, 24 and 13 glycopeptides could be enriched by ZIC®-HILIC and ProteoExtract® Glycopeptide Enrichment Kit, respectively. The results indicated that MoS2/Au-NP-L-cysteine nanocomposites have good adaptability and selectivity. In order to evaluate the batch-to-batch reproducibility, five batches of MoS2/Au-NP-L-cysteine nanocomposites were prepared. As shown in Fig. S4, Table S10 and Table S11, 31, 31, 34, 35 and 34 glycopeptides from IgG tryptic digests could be detected, respectively. In addition, 39, 36, 42, 42 and 36 glycopeptides from the mixture at a mass ratio of 1:2:4:4 of tryptic digests of HRP: RNase B: IgG: BSA could be detected (Fig. S5, Table S9 and Table S12), respectively. These exper-

Figure 4. MALDI-TOF MS spectra of (a), (b) and (c) were 0.67 pmol, 20 fmol and 10 fmol of IgG tryptic digest after enrichment by MoS2/Au-NP-L-cysteine, respectively. (e), (d) and (f) were 0.67 pmol, 50 fmol and 10 fmol of IgG tryptic digest after enrichment by ZIC®-HILIC, respectively.

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imental results indicated satisfying batch-to-batch reproducibility of MoS2/Au-NP-L-cysteine nanocomposites for glycopeptide enrichment.

Figure 6. MALDI-TOF MS spectra of HRP tryptic digest (0.5 µg). (a) direct analysis, (b) after enrichment by MoS2/Au-NPL-cysteine. Enrichment of glycopeptides from the proteins of HeLa cell exosomes. Inspired by the above advantages of MoS2/AuNP-L-cysteine nanocomposites for glycopeptide enrichment, such as good selectivity, large binding capacity, and high sensitivity, the MoS2/Au-NP-L-cysteine nanomaterials were further applied to enrich glycopeptides from complex biosamples. Exosomes, as the universal clinical specimen, have attracted increasing interest for the discovery of biomarkers of disease diagnosis. In this work, the glycopeptides from the glycoproteome profiling of HeLa cell exosomes have been analysed. Fifty micrograms of the tryptic digest of a protein mixture extracted from the exosomes of HeLa cell was analysed. As shown in Fig.S6, Table S13 and Table S16, 1359 peptides from 632 proteins were found to be N-glycosylated (Table S13), while 1129 peptides from 173 proteins were found to be O-glycosylated (Table S16) by MoS2/Au-NP-Lcysteine nanocomposites. In total, 1920 glycopeptides (Fig. S6c) from 775 glycoproteins (Fig. S6f) were enriched and identified from the tryptic digests of proteins extracted from exosomes of HeLa cells by using MoS2/Au-NP-L-cysteine nanocomposites in three repeated samples. For comparison, a total of 1591 glycopeptides (Fig. S6c) from 642 glycoproteins (Fig. S6f) were enriched from the tryptic digest of proteins extracted from exosomes of HeLa cells by using commercial ZIC®-HILIC for three experiments, in which 1098 peptides (Fig. S6a) from 509 proteins (Fig. S6d) were found to be Nglycosylated (Table S14), while 830 peptides (Fig. S6b) from 173 proteins (Fig. S6e) were found to be O-glycosylated (Table S17). Moreover, commercial ProteoExtract® Glycopeptide Enrichment Kit was chosen as a control enrichment material to enrich glycopeptides from the tryptic digest of proteins extracted from exosomes of HeLa cells. In total 724 glycopeptides (Fig. S6c) from 383 glycoproteins (Fig. S6f) were enriched from the tryptic digest of proteins extracted from exosomes of HeLa cells by using commercial ProteoExtract® Glycopeptide Enrichment Kit for three experiments, in which 262 N- and 620 O-glycosylated peptides from 175 N- and 241 Oglycosylated proteins were enriched and identified (Fig.S6, Table S15 and Table S18), respectively. The results indicated that the MoS2/Au-NP-L-cysteine nanocomposites exhibited better performance in N-glycoproteome analysis compared with commercial ZIC®-HILIC and ProteoExtract® Glycopeptide Enrichment Kit. Although MoS2/Au-NP-L-cysteine nanocomposites did not show a significant effect in O-glycoprotein enrichment compared with commercial ProteoExtract® Glycopeptide Enrichment Kit, it can also effectively

Figure 5. MALDI-TOF MS spectra of the mixture of IgG and BSA tryptic digest with (a), (b), (c) and (d) were a mass ratio of 1:1 (3.34 pmol IgG digest), 1:100 (100 fmol IgG digest), 1:200 (50 fmol IgG digest) and 1:500 (20 fmol IgG digest) after enrichment by MoS2/Au-NP-L-cysteine, respectively. (e), (f) and (g) were a mass ratio of 1:1 (3.34 pmol IgG digest), 1:10 (500 fmol IgG digest), 1:100 (100 fmol IgG digest) and 1:500 (20 fmol IgG digest) after enrichment by commercial ZIC®-HILIC, respectively.

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Analytical Chemistry enrich and identify O-glycoprotein, indicating that MoS2/AuNP-L-cysteine nanocomposites exhibited higher sequence coverages of O-glycoproteins although the numbers of Oglycoproteins are equal to or less than those enriched with commercial ZIC®-HILIC or a ProteoExtract® Glycopeptide Enrichment Kit. In addition, as shown in Table S22, the selectivity of glycopeptide enrichment (the number of both types of glycopeptides divided by the total number of identified peptides) was 52.46%, which indicated that the MoS2/Au-NP-Lcysteine nanocomposites have higher specificity compared with commercial ZIC®-HILIC (selectivity, 48.48%) and a ProteoExtract® Glycopeptide Enrichment Kit (selectivity, 26.49%). The detailed information on the identified glycoproteins is listed in Table S13~S21. Futhermore, in order to determine extraction quantity, nanodrop spectrophotometer was used to determine the amount of enriched peptides.45 As shown in Table S23, the average extraction quantity of glycopeptides from 50 µg tryptic digest of proteins extracted from exosomes of HeLa cells were 3.13%±0.79% (n=3), 2.95%±0.73% (n=3) and 2.52%±1.06% (n=3) with MoS2/AuNP-L-cysteine nanocomposites, Commercial ZIC®-HILIC and a ProteoExtract® Glycopeptide Enrichment Kit, respectively. Taken together, the experimental results showed that MoS2/Au-NP-L-cysteine nanocomposites exhibited more identification number, higher selectivity and higher extraction yield of glycopeptide compared with commercial ZIC®-HILIC and a ProteoExtract® Glycopeptide Enrichment Kit in glycoproteomic analysis of exosomes of HeLa cells. This suggested that MoS2/Au-NP-L-cysteine nanocomposites-based enrichment method provided an alternative approach to effectively enrich and identify both N- and O-linked glycopeptides. Besides, because of their respective advantages of the different approaches (MoS2/Au-NP-L-cysteine nanocomposites-, commercial ZIC®-HILIC- and a ProteoExtract® Glycopeptide Enrichment Kit-based enrichment methods) in glycoproteomic analysis, the combination of these different approaches could be used to improve the sensitivity and coverage in-depth analysis of glycoproteomes when applied to complex bio-samples.

AUTHOR INFORMATION Corresponding Author * Yehua Shen Email: [email protected] Phone: +86-029-883 * Yangjun Zhang Email: [email protected] Phone: +86-010-61777110 CONFLICTS OF INTEREST

There are no conflicts to declare. ACKNOWLEDGMENT This work was supported by National Key R&D Program of China (2017YFA0505002 and 2017YFC0906703), the National Natural Science Foundation of China (21675125 and 21606181), the National Key Program for Basic Research of China (2013CB911204 and 2016YFA0501403), the China Postdoctoral Science Foundation (2017M613331). REFERENCES (1) Witze, E. S.; Old, W. M.; Resing, K. A.; Ahn, N. G. Nature methods 2007, 4, 798. (2) Palaniappan, K. K.; Bertozzi, C. R. Chemical reviews 2016, 116, 14277-14306. (3) Ohtsubo, K.; Marth, J. D. Cell 2006, 126, 855-867. (4) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science 2001, 291, 2370-2376. (5) Hakomori, S.i. Cancer Res 1985, 45, 2405-2414. (6) Pinho, S. S.; Reis, C. A. Nature reviews. Cancer 2015, 15, 540. (7) Zhang, Y.; Jiao, J.; Yang, P.; Lu, H. Clinical proteomics 2014, 11, 18. (8) Woo, C. M.; Iavarone, A. T.; Spiciarich, D. R.; Palaniappan, K. K.; Bertozzi, C. R. Nature methods 2015, 12, 561-567. (9) Dell, A.; Morris, H. R. Science 2001, 291, 2351-2356. (10) Olsen, J. V.; Mann, M. Molecular & Cellular Proteomics 2013, 12, 3444-3452. (11) Kaji, H.; Saito, H.; Yamauchi, Y.; Shinkawa, T.; Taoka, M.; Hirabayashi, J.; Kasai, K.i.; Takahashi, N.; Isobe, T. Nature biotechnology 2003, 21, 667. (12) Zhang, H.; Xiao-jun, L.; Martin, D. B.; Aebersold, R. Nature biotechnology 2003, 21, 660. (13) Mysling, S.; Palmisano, G.; Højrup, P.; Thaysen-Andersen, M. Analytical chemistry 2010, 82, 5598-5609. (14) Lin, H.; Ou, J.; Zhang, Z.; Dong, J.; Wu, M.; Zou, H. Analytical chemistry 2012, 84, 2721-2728. (15) Sun, N.; Wang, J.; Yao, J.; Deng, C. Analytical chemistry 2017, 89, 1764-1771. (16) Zhu, H.; Li, X.; Qu, J.; Xiao, C.; Jiang, K.; Gashash, E.; Liu, D.; Song, J.; Cheng, J.; Ma, C. Analytical and bioanalytical chemistry 2017, 409, 511-518. (17) Jiao, F.; Gao, F.; Wang, H.; Deng, Y.; Zhang, Y.; Qian, X.; Zhang, Y. Scientific Reports 2017, 7, 6984:1-7. (18) Jiao, F.; Gao, F.; Wang, H.; Deng, Y.; Zhang, Y.; Qian, X.; Zhang, Y. Analytica Chimica Acta 2017, 970, 47-56. (19) Zhang, Y.W.; Li, Z.; Zhao, Q.; Zhou, Y.L.; Liu, H.-W.; Zhang, X.-X. Chemical communications 2014, 50, 11504-11506. (20) Ma, W.; Xu, L.; Li, Z.; Sun, Y.; Bai, Y.; Liu, H. Nanoscale 2016, 8, 10908-10912. (21) Ma, W.; Xu, L.; Li, X.; Shen, S.; Wu, M.; Bai, Y.; Liu, H. ACS Applied Materials & Interfaces 2017, 9, 19562-19568 . (22) Wu, H.; Yang, R.; Song, B.; Han, Q.; Li, J.; Zhang, Y.; Fang, Y.; Tenne, R.; Wang, C. ACS nano 2011, 5, 1276-1281. (23) Liu, T.; Shi, S.; Liang, C.; Shen, S.; Cheng, L.; Wang, C.; Song, X.; Goel, S.; Barnhart, T. E.; Cai, W. ACS nano 2015, 9, 950-960. (24) Nezhad, M. R. H.; Fahimi-Kashani, N.; Rashti, A.; Mahdavi, V. Analytical Methods 2017, 9, 716-723

CONCLUSIONS In this study, MoS2/Au-NP-L-cysteine nanocomposites, a novel kind of ZIC-HILIC material, were successfully prepared. This novel material showed great advantages in identifying many low-abundance glycopeptides in both model glycoprotein tryptic digest and the glycoproteome profiling of the HeLa cell exosomes, owing to the virtues of a large surface area, good biocompatibility, ultra-high hydrophilicity and easy preparation of this nanomaterial. The nanomaterials showed high specificity, extremely high detection sensitivity, large binding capacity, high recovery and satisfying batch-to-batch reproducibility in glycopeptide enrichment. The outstanding glycopeptide enrichment ability makes this nanomaterial a promising candidate for glycoproteome analysis and offers great potential for the discovery of biomarkers for early disease diagnosis. Additionally, this work also introduces a new field for the applications of MoS2 in proteomics studies. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

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