High Strength and Hydrophilic Chitosan Microspheres for the

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High Strength and Hydrophilic Chitosan Microspheres for the Selective Enrichment of N-Glycopeptides Xiaomei He, Xichao Liang, Xi Chen, Bifeng Yuan, Ping Zhou, Lina Zhang, and Yu-Qi Feng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01283 • Publication Date (Web): 22 Aug 2017 Downloaded from http://pubs.acs.org on August 24, 2017

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Analytical Chemistry

High Strength and Hydrophilic Chitosan Microspheres for the Selective Enrichment of N-Glycopeptides Xiao-Mei He,†1 Xi-Chao Liang,†2 Xi Chen,3 Bi-Feng Yuan,1 Ping Zhou,2 Li-Na Zhang,*2 Yu-Qi Feng*1

1

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of

Education), Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China 2

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P.R.

China. 3

Wuhan Institute of Biotechnology, Wuhan 430072, P.R. China.

†

These authors contributed equally to this work

Corresponding author: *

Yu-Qi Feng, Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry

of Education), Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China. Tel: +86-27-68755595; Fax: +86-27-68755595; E-mail: [email protected] *

Li-Na Zhang, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan

430072,

P.R.

China.

Tel:

+86-27-87219274;

Fax:

[email protected]

1

ACS Paragon Plus Environment

+86-27-87219274;

E-mail:


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Abstract Protein glycosylation is an important post-translational modification that plays a crucial role in many biological processes. Due to the low abundance of glycoproteins and high complexity of clinical samples, the development of methods to selectively capture glycoproteins/glycopeptides is crucial to glycoproteomics study. In this work, a kind of highly cross-linked chitosan microspheres (CSMs) was prepared using epichlorhydrine as cross-linker from chitosan solution in alkaline/urea aqueous system. The results showed that CSMs had high amino groups content, large surface area, mesoporous structure, good acidic resistance and high strength by various tests. On the basis of hydrophilic interaction between the polar groups (amino groups and hydroxyl groups) on CSMs and glycan moieties on glycopeptides, the prepared CSMs were applied to specific capture of N-glycopeptides from standard protein digests and complex biological samples (body fluids and tissues). The CSMs exhibited high selectivity (HRP/BSA = 1:100), good sensitivity (4.5 ×10−10 M of HRP), good recovery yield (74.9% to 106.4%) and high binding capacity (100 mg g-1) in glycopeptides enrichment. Due to the excellent performance in glycopeptides enrichment, CSMs were applied to selectively enrich N-glycopeptides from tryptic digests of human serum and rat brain followed by nanoLC-MS/MS analysis. We identified 194 and 947 unique N-glycosylation sites from 2 µL of human serum and 0.1 mg of rat brain, respectively. Additionally, the extraction time of our method was much shorter than the previously reported methods. Therefore, the fabricated CSMs with desirable properties will find broad application in large-scale and in-depth N-glycoproteome analysis.

2


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Keywords: chitosan microspheres; hydrophilic interaction chromatography; N-glycopeptides; human serum; rat brain

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Introduction Glycosylation, one of the most important and complex post-translational modifications of proteins, plays a crucial role in various biological processes, including protein folding, cell communication, signal transduction and immune response.1,2 Aberrant glycosylation is related to the initiation and progression of tumors, and the majority of the discovered cancer biomarkers are proved to be glycosylated proteins or peptides.3 Therefore, intensive efforts have been devoted to the development of glycoproteome identification techniques. In the recent years, mass spectrometry (MS) has been proved to be the most powerful tool for glycoproteme analysis due to its high sensitivity and high-throughput, and capacity to analyze differences in disease-assosiated glycoforms.1,4 However, the low abundance of glycoproteins in high matrix complexity of clinical samples (e.g., body fluids and tissues), the poor ionization of glycopeptides in MS detection and the heterogeneity of glycoforms make the MS-based glycoproteome analysis extremely difficult. Therefore, enrichment of the target glycopeptides from the complex samples prior to MS detection is a prerequisite of successful analysis of glycoproteome. To date, the general adopted strategies for glycopeptide enrichment are mainly based on hydrazine chemistry,5 hydrophilic interaction chromatography (HILIC),6-8 boronic acid-based covalent

capture

technology,9-12

lectin

affinity

chromatography,13

and

chelation

interaction.14-16 Among them, the HILIC-based enrichment method by utilizing the hydrophilicity of glycopeptide has gained increasing popularity because of its good reproducibility, reversible for the glycopeptides alterations and MS compatibility.17 Chitosan is a kind of polysaccharide derived from the deacetylation of chitin, which has attracted considerable attention in the biomedical domain due to its inherent advantages such 4


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as biocompatibility, non-toxicity, biodegradability, as well as antibacterial activity.18-21 In the recent years, the use of carbohydrate-based ligands, such as glucose,22 maltose,6,23 agarose24, cellulose25,26 and cyclodextrins27 as hydrophilic groups for glycopeptides enrichment is an emerging trend. Like other saccharides, chitosan with abundant amount of polar groups (amino groups and hydroxyl groups) has the ability to capture glycopeptides through interaction with the glycan moieties by hydrogen bonding. However, the poor mechanical properties and acid instability of chitosan seriously restrict its practical applications. In this regard, some researchers investigated the assembly of chitosan on solid supports for enrichment of glycopeptides. For instance, Xiong et al. prepared multilayer polysaccharide coated magnetic nanoparticles for glycopeptide enrichment using layer-by-layer assembly of hyaluronan and chitosan.28 However, the synthesis process was tedious and time-consuming, and the cross-linking of carboxyl groups on hyaluronan and amino groups on chitosan might compromise its hydrophilicity. Thereupon, the same group developed a one-pot solvothermal method for the preparation of magnetic colloidal nanocrystal coated with chitosan for glycopeptides enrichment.29 Although the proposed method was simple, Fe3O4 was not able to be covered completely by chitosan, which could result in nonspecific adsorption from the exposed Fe3O4. Therefore, it is essential to establish a simple and facile approach to prepare high strength chitosan material without destroying the hydrophilicity of chitosan. In this work, we prepared porous chitosan microspheres (CSMs) with high strength and good acidic resistance using epichlorhydrine (ECH) as cross-linker from chitosan solution in alkaline/urea aqueous system via emulsification procedure. Here, the chitosan was dissolved due to the destruction of intermolecular hydrogen bonds in alkaline aqueous system rather 5



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than protonation of the amino groups in acidic solution.30 As the cross-linking of chitosan was based on the etherification between hydroxyl groups on chitosan and ECH, the hydrophilicity of chitosan could be well preserved, which was different from traditional chitosan materials prepared by cross-linking in chitosan acidic solution with glutaraldehyde.30 Afterwards, the prepared CSMs with good biocompatibility, high porosity and high surface area, excellent hydrophilicity and solvent resistance were applied to specific capture of N-glycopeptides from complex biological samples including human serum and rat brain.

2. Experimental Section Materials Commercial grade chitosan (with a deacetylation degree of 89%) was purchased from Ruji Biotechnology Co., Ltd. (Shanghai, China) and used without further purification. Epichlorohydrin (ECH), isooctane and Span 80 were all of analytical grade supplied by Sinopharm Chemical Reagent Co., Ltd (Beijing, China) and were used without further purification. Trifluoroacetic acid (TFA), 2,5-dihydroxybenzoic acid (2,5-DHB), horse radish peroxidase (HRP), chicken avidin, human immunoglobulin G (IgG), bovine serum albumin (BSA), bovine fetuin, Sepharose CL-6B (45~165 µm), formaldehyde (37% solution in H2O) and formaldehyde-D2 (20% solution in D2O) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PNGase F was purchased from New England Biolabs (Ipswich, MA). HPLC grade acetonitrile (ACN) was purchased from Fisher Scientific (Pittsburgh, PA, USA). Sequencing grade trypsin was purchased from Promega (Madison, WI, USA). Purified water was obtained with a Milli-Q apparatus (Millipore, Bedford, MA, USA). Human serum 6


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samples were collected from healthy people and obtained from The Hospital of Wuhan University according to the standard clinical procedures. The utilization of human serum complied with guidelines of Ethics Committee of the Institute, and all participants gave their informed consent. Sprague dawley (SD) male rat (about 220 g in weight) was purchased from Hubei provincial center for disease control and prevention (Wuhan, Hubei, China). Preparation of chitosan microspheres The chitosan microspheres (CSMs) were fabricated via a sol-gel transition method by conventional emulsion process. In detail, 4 g of CS powder was dispersed in 96 g of alkaline aqueous solvent of LiOH, KOH, urea, and water (4.5:7:8:80.5, by weight) to obtain a suspension. Subsequently, the resulted suspension was frozen at -40 oC for 4 h, and then the frozen solid was fully thawed by stirring vigorously at room temperature. The freezing-thawing cycle was repeated twice to obtain a transparent CS solution with concentration of 4 wt%. Then, 2 mL of ECH as a cross-linker was added into the CS solution and stirred at -20 oC for 2 h to obtain a homogeneous pre-gel solution. The obtained pre-gel solution was centrifuged at 7000 rpm for 10 min at 0 oC to remove air bubbles. After that, the CS pre-gel solution was dropped into 500 mL of flask containing 250 g of isooctane and 20 mL of Span 80 under vigorous stirring at 1000 rpm in the ice-water bath for 30 min to form a colloidal suspension. After removing the ice-water bath, the suspension was completely gelled after stirring at room temperature for 3~4 h to form the CS microspheres (CSMs). The obtained CSMs were transferred into 1 L solution of ethanol/water (7:3 by volume) and stirred for 1 h. Finally, the CSMs were washed with ethanol and deionized water repeatedly to remove the residual reagent, and then frozen-dried for further characterization. 7



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Characterization of chitosan microspheres The morphology and size of CSMs were characterized using the field emission scanning electron microscopy (FE-SEM, Zeiss, SIGMA) and the optical microscopy (OD500M, Sunny Optical Technology Co., Ltd. China). Surface area and pore size distribution were evaluated using nitrogen adsorption (Micromeritics, AsAp2020, U.S.A.). The specific surface area was calculated from the nitrogen adsorption isotherm using the Branauer-Emmett-Teller (BET) equation and the pore size distribution using the Barrett-Joyner-Halenda (BJH) model. The swelling behaviors of CSMs were measured at different pH ranging from 1 to 13, which were determined using a FE-20K pH meter (Mettler-Toledo, Ohio, U.S.A); the swelling ratio was calculated as: swelling ratio = (Ws-Wd)/ Wd, where Ws and Wd were the weights of the swollen and dried microspheres, respectively. Compressive measurements were performed on CSMs with the Fourier transform infrared spectroscopy (FT-IR, NICOLET 5700, Thermo Nicolet) compression mold using a universal testing machine (CMT 6503, Shenzhen, SANS, China) according to ISO527-3-1995 (E) at speed of 1 mm min-1. The load and displacement data were collected during the experiments. The structures of CSMs were characterized by FT-IR and wide-angle X-ray diffraction (XRD, D8-Advance, Bruker). Solid-state 13C NMR spectra with cross polarization/magic angle spinning (CP/MAS) of the samples were carried out on a 300 MHz NMR spectrometer (Bruker Advance III) at ambient temperature. The swelling behaviors of CSMs were measured at different pH adjusted by 0.1 M HCl or 0.1 M NaOH aqueous solution, ranging from 1 to 13, which were determined by using a FE-20K pH meter (Mettler-Toledo, Ohio, U.S.A). The ionic strength of the pH solution was controlled to be 0.1 M by adding an appropriate amount of NaCl solution. The swelling temperature was set as 37 8


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o

C to simulate body temperature for exploring potential biomedical application. The swelling

ratio was calculated as Swelling ratio = (Ws-Wd)/Wd

(1)

Where Ws and Wd are the weights of the swollen and dried microspheres, respectively. The content of amino groups of CSMs was determined by the two-abrupt-charge potentiometric titration method [6]. In detail, 0.2 g CSMs were dispersed under magnetic stirring in 20 mL of 0.1 M HCl for 12 h to make the amino group on microspheres fully protonated. Measurements were performed with a solution of 0.1 M NaOH by using a FE-20K pH meter (Mettler-Toledo, Ohio, U.S.A). The content of amino groups of CSMs was calculated as follows: ∆V×CNaOH ×10-3 ×16 (2) Amino group content = m Where ∆V is the volume of NaOH consumption between the two abrupt changes of pH, CNaOH is the concentration of the NaOH solution, m is the dry weight of CSMs sample, 16 is the related molecular weight of amino groups. Glycopeptides enrichment by chitosan microspheres CSMs (0.3 mg) were dispersed in loading buffer (90% ACN, 9% H2O, 1% TFA, by volume) containing a certain amount of HRP digests, avidin digests or IgG digests. After vortex for 2 min, the supernatant was removed after centrifugation at 6000 rpm (Kelin bell, LX-400) for 5 s. Then, the glycopeptides-absorbed CSMs were washed twice with loading buffer. Finally, the adsorbed glycopeptides were eluted from CSMs with 0.1% TFA aqueous solution (100 µL) for 1 min. The whole procedure can be accomplished within 5 min. Then the eluted solution was lyophilized to dryness. Finally, 1.2 µL of matrix solution (mixture of 9



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12 mg/mL 2,5-DHB in 50% ACN, 49.9% H2O, 0.1% TFA, by volume) was introduced into the residue and the mixture was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For the glycopeptides enrichment from fetuin digests, the loading buffer and desorption solution were 90% ACN-0.1% TFA aqueous solution and 5% TFA aqueous solution, respectively. The eluted solution was collected for analysis by TripleTOF 5600+ mass spectrometry. For the glycopeptides enrichment from the tryptic digests of human serum or rat brain, the sample loading and washing processes were the same as that for the glycopeptides enrichment from HRP digests. Then, the trapped glycopeptides were eluted twice with 0.1% TFA aqueous solution (100 µL) for 1 min, and the eluted solution was evaporated to dryness. The obtained glycopeptides were redissolved in 20 µL of NH4HCO3 (10 mM, pH 7.5), and 1000 units of PNGase F were added. Subsequently, the mixture was incubated at 37 °C for overnight to remove the glycan moieties. Finally, the mixture was desalted with Ziptip C18, and used for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Recovery yield test of chitosan microspheres for glycopeptide enrichment The recovery yields of glycopeptides were also investigated by stable isotope dimethyl labeling according to previously described strategy.6,31 Briefly, two equivalent protein digests were firstly labeled with light (+28 Da) and heavy dimethyl isotopes (+32 Da), respectively. The light-tagged digests were enriched with CSMs according to the above-mentioned procedure, and the resulting elution was spiked into the heavy-tagged digests. The combined mixture was re-enriched with CSMs, and the eluted fraction was analyzed by 10


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MALDI-TOF-MS. The recovery yields were calculated by peak intensity ratios of light-labeled glycopeptides/heavy-labeled glycopeptides.

Results and discussion Morphology and structure properties of chitosan microspheres In the previous work, a high strength chitosan hydrogel was constructed using ECH as cross-linker in the chitosan alkaline solution without depressing the desirable properties of chitosan (CS).30 The proposed chitosan hydrogel constructed from the alkaline solution system exhibits excellent mechanical properties, unique nanofibrous architecture, good biocompatibility and hydrophilicity, which has the potential to serve as HILIC material. However, the blocky structure and low surface area of chitosan hydrogel restrict its applications in sample preparation. In this work, porous chitosan microspheres (CSMs) were prepared from the chitosan alkaline solution via crosslinking reaction and emulsification procedure (Figure 1). Firstly, the solid-state

13

C NMR spectra were applied to study the

mechanism in crosslinking reaction. Figure S1 showed the solid-state

13

C NMR spectra of

CSMs and pristine CS powder. Obviously, CS powder and CSMs exhibited the similar

13

C

NMR spectra and the assignment of the observed signals to various types of carbons was demonstrated. However, it is well worth noting that the peak at 61.43 ppm for C6 of CS powder displayed a large loss of resolution in CSMs and the shoulder peak appeared at about 61.51 ppm, indicating the crosslinking reaction had occurred on hydroxyl groups on C6, in agreement with the results of crosslinking reaction between CS and ECH.32-36 The 13C NMR results confirmed that cross-linking reaction occurred, owing to the etherification reaction 11



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between ECH and hydroxyl groups on CS, and the proposed schematic representation for the crosslinking reaction was shown in Figure 1d. To examine whether the amino groups on CS were also involved in the cross-linking reaction, the content of amino groups of CSMs and pristine CS powder were measured by two-abrupt-charge potentiometric titration method.37 Potentiometric titration of CS powder and CSMs gave a curve that had inflexion points (Figure S2a). The first and second inflexion points were the equivalence points of the titration of excessive HCl and the titration of protonated amino groups, respectively, and the accurate points could be obtained from Figure S2b. The content of amino groups of CSMs and CS powder were calculated to be 0.07209 and 0.08681, respectively. The theoretical amount of amino groups on chitosan is 0.0994 with a deacetylation degree of 100%. The titrated results showed that CSMs contained a large amount of amino groups, suggesting that most of the amino groups on CS were not involved in crosslinking reaction. Therefore, based on the above results, the CSMs fabricated in alkali/urea aqueous system retained a large number of amino groups and exhibited excellent hydrophilicity, which was crucial for our further application. As shown in Figure 2, CSMs exhibited a spherical shape with mean diameter of 40 µm, and had narrow size distribution as well as smooth surface. The surface of the CSMs displayed nano-size pore microstructure. To further characterize the porous structure of CSMs, a nitrogen adsorption-desorption measurement on CSMs was carried out (Figures 3a and 3b). The surface area and the corresponding nano-scaled pore size of the CSMs were determined from N2 adsorption-desorption to be 191 m2 g-1 and 10 nm, suggesting the high surface area and characteristic mesoporous structure of CSMs. The results indicated the potential 12


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application of CSMs as an adsorbent for sample preparation. The structural stability and mechanical properties of the microspheres are important for their practical applications. The swelling ratios of CSMs at different pH values were shown in Figure 3c. It was noted that the CSMs exhibited swelling behavior when pH < 4 due to the existence of the strong electrostatic repulsion between the protonated amino groups in the chitosan chains while did not dissolve in the acidic environment. We reasoned that the cross-linking of chitosan in the alkaline condition was based on the etherification between hydroxyl groups on chitosan and ECH, leading to the good acidic resistance of CSMs. Moreover, the compressive test was performed on the CSMs with the FT-IR infrared compression mold to evaluate the mechanical properties of CSMs (Figure S3). The typical compressive stress-strain curve (Figure 3d) indicated the high strength of the CSMs, which could be attributed to the unique interconnected 3D network architecture in the microspheres. These results supported the favorable structure stability of CSMs, which will play a dominant role in its practical applications. The structures of CSMs were characterized by FT-IR spectrum and XRD pattern. FT-IR spectrum of CSMs was shown in Figure S4a. The amide peaks of chitosan at 1652 and 1571 cm-1 were ascribed to vibrations of amide I and amide II band, respectively, and the peak at 1083 cm-1 corresponded to the vibration of glycosidic bonds. Moreover, other characteristic peaks of CS at 3429 cm-1 (O-H stretch and N-H stretch, overlap), 2923 cm-1 (C-H stretch), 1158 cm-1 (bridge-O- stretch) were also identified. The XRD pattern of CSMs was shown in Figure S4b. Two crystalline diffraction peaks were observed in CSMs, indexed as (020) and (110) lattice diffraction of CS, further indicating that the CSMs was constructed from CS.35,36 13



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Evaluation of the property of chitosan microspheres toward N-glycopeptides The selective recognition of CSMs to glycopeptides was based on the hydrophilic interaction between the polar groups (amino groups and hydroxyl groups) on CSMs and glycan moieties on glycopeptides. To achieve the best enrichment efficiency of CSMs, several conditions were optimized using tryptic digests of HRP (a standard glycoprotein). Before enrichment, non-glycopeptides peaks with high intensities dominated the spectrum and only 6 glycopeptides were detectable with low signal-to-noise ratios (S/N) (Figure 4a). However, after enrichment with CSMs, 24 glycopeptides (detailed information of detected glycopeptides is listed in Table S1) with enhanced S/N could be observed and almost all signals of non-glycopeptides were removed (Figure 4b), indicating the good affinity of CSMs toward glycopeptides. Four representative glycopeptides structures from HRP were confirmed based on their MALDI-TOF/TOF tandem mass spectra (Figure S5). The MS/MS spectra of intact glycopeptides also showed that they predominantly generated ions that were due to glycan fragmentations while few peptide backbone fragmentations were observed. Meanwhile, no glycopeptides could be detected in sampling eluate (Figure 4c), which suggested the extraction equilibration of glycopeptides could be achieved within 2 min. The extraction time of this work was much shorter than the previous HILIC-based methods,6,7,28,29,38 indicating the fast mass transfer property of CSMs due to its high porosity and good permeability. Cross-linking can improve the acidic resistance of chitosan materials in harsh environment. In this work, ECH was used to cross-link chitosan to prepare CSMs under alkaline condition without destroy its hydrophilicity, which was different from traditional 14


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methods using glutaraldehyde (GA) as cross-linking agent in acidic solvent system.30 To illustrate the importance of hydrophilicity, CSMs were further cross-linked with GA solution (2.5 %), and then used for the selective enrichment of glycopeptides from HRP digests. As shown in Figure 4d, only 2 glycopeptides along with many abundant non-glycopeptides were observed after enrichment with GA cross-linked CSMs. Therefore, the proposed CSMs could be employed for specific capture of glycopeptides, and the high selectivity and affinity of CSMs toward glycopeptides was from the inherent hydrophilicity of chitosan. The enrichment specificity of CSMs toward glycopeptides was further evaluated using HRP digests with different interference levels of BSA digests (HRP: BSA= 1:10 and 1:100). With the addition of BSA digests, the signals of non-glycopeptides dramatically enhanced and the identification of glycopeptides became impossible by direct analysis (Figures 5a and 5c). However, 23 and 21 glycopeptides could be distinctly detected after CSMs enrichment with the molar ratios of HRP and BSA being 1:10 (Figure 5b) and 1:100 (Figure 5d), respectively, which showed significant improvement of the selectivity for glycopeptides than many reported materials.6,7,9,29,38 The results indicated the excellent selectivity of CSMs toward glycopeptides even in the presence of a large amount of interfering non-glycopeptides. For comparison, a commonly used HILIC material, Sepharose CL-6B, was also applied to the selective enrichment of glycopeptides. The extraction conditions for Sepharose CL-6B were according to previous reports23 with slight modification. As shown in Figure S6, only 12 and 4 glycopeptides along with lots of non-glycopeptides were observed after Sepharose CL-6B enrichment from HRP digests and the mixture of HRP and BSA digests (HRP: BSA= 1:10), respectively. The results showed a superior selectivity of CSMs toward glycopeptides than 15



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Page 16 of 34

commercially available Sepharose CL-6B, which might be due to the better hydrophilicity and larger surface area of CSMs than Sepharose CL-6B. The sensitivity of this approach was investigated with different concentrations of the HRP digests. The signal intensities of glycopeptides gradually weakened with the decrease in concentration of HRP digests. When the concentration of HRP digests was as low as 4.5 ×10−10 M, one glycopeptide with S/N at 12.1 could still be well observed after enrichment with CSMs (Figure S7), demonstrating the high detection sensitivity of this strategy. To validate the unbiased enrichment of N-glycopeptides by CSMs, chicken avidin contains high-mannose and hybrid-type N-glycans, and human IgG carrying complex-type N-glycans were employed. Direct analysis of avidin and IgG digests showed that the signals of glycopeptides were severely suppressed by non-glycopeptides (Figures 6a and 6d). After enrichment with CSMs (Figures 6b and 6e), 23 and 24 glycopeptides with good resolution were identified in avidin and IgG digests, respectively (detailed information of detected glycopeptides is listed in Tables S2 and S3). Some representative glycopeptides structures from avidin and IgG were confirmed based on their MALDI-TOF/TOF tandem mass spectra (Figures S8 and S9). Additionally, the eluted fractions from CSMs enrichment were deglycosylated by PNGase F (a N-glycosidase) and two deglycosylated peptides were detected in each mass spectrum (Figures 6c and 6f), which demonstrated that the peaks in Figure 6b and Figure 6e were N-glycopeptides. However, the result of avidin digests after PNGase F treatment was not consistent to the previous works.28,39 As far as we know, chicken avidin has a single glycosylation site at Asn40, thus there should be only one rather than two deglycosylated

peptides

(m/z=

1837) in Figure

6c.

To explore

16


this

anomaly,

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MALDI-TOF/TOF tandem mass spectra of the two deglycosylated peptide (m/z= 1789 and 1837) from avidin were used. As shown in Figure S10, the MS/MS spectra of two deglycosylated peptides were well matched in the m/z range of 200-850, while a 48 Da mass difference was observed in y ions (from y9 to y15) in the m/z range of 850-1600. The results indicated that the 48 Da mass loss in the peptide of WTNDLGSNMTIGAVNSR occurred at methionine (M), and the 48 Da mass loss might be CH3SH. Meanwhile, 8 glycopeptides in connection with the peptide of WTNDLGSN(M-48)TIGAVNSR were also observed in the mass spectrum (Figure 6b). The recovery yields of glycopeptides from avidin and IgG with a range of glycoforms were also investigated by stable isotope dimethyl labeling.6,31 As shown in Table S4, the recovery yields of representative glycopeptides with diffenent glycoforms had no significant difference, which ranged from 74.9% to 106.4%. The results indicated the good recovery and no bias of CSMs for the selective enrichment of glycopeptides. As mentioned above, CSMs is positively charged when pH < 4 due to the protonated amino groups, therefore, CSMs should have high affinity towards acidic sialylated glycopeptides owing to electrostatic interaction. To verify this, bovine fetuin was chosen as a model sialylated glycoprotein. Direct analysis of fetuin digests showed that the signals of glycopeptides were severely suppressed by non-glycopeptides (Figure 7a). After enrichment with CSMs, a total of 35 N-glycopeptides signals with enhanced intensity were identified, and all of them contained sialic acid termination (Figure 7b). Detail information of detected glycopeptides is listed in Table S5. Four representative glycopeptides structures from fetuin were confirmed based on their nanoESI-MS/MS tandem mass spectra (Figure S11). The results demonstrated the feasibility of CSMs for the enrichment of sialylated glycopeptides, 17



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further verifying the good universality of CSMs for glycopeptides enrichment. The binding capacity of CSMs toward glycopeptides was investigated by adding different amounts of CSMs (10-100 µg) to a fixed amount of IgG digests (3 µg). After enrichment, the eluted fraction from CSMs (0.5 µL from 10 µL total) was analyzed by MALDI-TOF-MS. When the amount of CSMs added was 30 µg, the intensities of six selected glycopeptides reached the maximum and the binding capacity of CSMs was calculated to be 100 mg g-1 (Figure S12), which was higher than some HILIC materials such as Fe3O4-DA-Maltose

(43

mg

g-1),6

Fe3O4@CS

MCNCs

(17.5

mg

g-1)29

and

MCM-41-APTES-CPB (40 mg g-1).40 The large binding capacity is due to high amount of polar groups on CSMs and strong multivalent hydrophilic interactions between CSMs and glycopeptides. N-Glycopeptides enrichment from tryptic digests of human serum To illustrate the feasibility of CSMs in large-scale N-glycoproteome analysis from complex biological samples, tryptic digests of human serum were applied for investigation. After incubation with the CSMs, the glycopeptides in tryptic digests of human serum were enriched, followed by elution, deglycosylation, and then analysis with LC-MS/MS. Combined with the mass increment of 0.98402 Da of asparagine (N) transforming into aspartic acid (D), the identification of N-glycosylation sites as well as N-glycopeptides could be clearly and definitely realized by the existence of N-glycosylation consensus motif (N-X-T/S/C, X ≠ P). A total of 194 unique N-glycosylation sites from 104 glycoproteins were identified from 2 µL of human serum (detailed information of detected N-glycopeptides is listed in Table S6). According to the Swiss-Prot and TrEMBL databases, 39 glycosylation sites were newly 18


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identified in the human serum. Compared with some previous works using 5-20 µL of human serum,41-45 our proposed method exhibited comparable performance in N-glycoproteome analysis with only 2 µL of human serum. The results indicated the feasibility of the proposed method for the N-glycoproteome profiling of complex biological samples. N-Glycopeptides enrichment from tryptic digests of rat brain Inspired by the aforementioned results, CSMs were further applied to in-depth N-glycoproteome analysis from more-complex tissue sample, rat brain. As we know, a series of biological processes in brain including the nervous system development, synaptic transmission, learning, memory, and processing of sensory information are associated with glycosylation.46 After treated with CSMs, 947 unique N-glycosylation sites corresponded to 466 glycoproteins were identified from 0.1 mg of rat brain (detailed information of detected N-glycopeptides is listed in Table S7). According to the Swiss-Prot and TrEMBL databases, 505 glycosylation sites were newly identified in the rat brain. The Website based programs motif-X47 and Weblogo48 were used to characterize the motif composition of identified N-glycosylation sites and create relative frequency plots, respectively. Figure 8a showed the distributions of the three motifs at 59.1% (N-X-T), 38.2% (N-X-S), and 2.7% (N-X-C), which were in accordance with the previous reports.49,50 The number of identified N-glycosylation sites in each protein was also investigated. As shown in Figure 8b, 53.5% and 22.6% of all glycoproteins were detected with a single N-glycosylation site and two N-glycosylation sites, respectively, and the average degree of N-glycosylation was 2.0. The results indicated the good performance of the CSMs in large-scale and in-depth N-glycoproteome analysis.

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Conclusion In the current study, highly cross-linked CSMs with high strength and good acidic resistance were prepared in alkaline/urea aqueous system via emulsification procedure. The CSMs that contain high surface area and mesoporous structure exhibited high selectivity, good sensitivity and high binding capacity as well as good universality in N-glycopeptides enrichment from HRP digests, avidin digests, IgG digests, fetuin digests and complex biological samples. The good performance was ascribed to the merits of CSMs, including excellent hydrophilicity, good biocompatibility, high surface area and abundant amount of affinity sites. In addition, the extraction time of this work was much shorter than the previous HILIC-based methods, indicating the fast mass transfer property of CSMs due to its high porosity and good permeability. Taken together, CSMs were proved to be a good HILIC adsorbent and the proposed method provided a new option for comprehensive large-scale N-glycoproteome analysis in complex samples. Last but not the least, CSMs equipped with a large number of highly reactive amino groups can be easily modified for further applications.

Acknowledgment We thank Prof. Xin Liu and Dr. Chang Wang at Huazhong University of Science and Technology, for their generous providing of MALDI-TOF-TOF MS and LC-MS/MS, and their valuable suggestions for revising this paper. Financial support is gratefully acknowledged from the National Basic Research Program of China (973 Program) (2013CB910702) and the National Natural Science Foundation of China (21475098, 31670373, 21635006). 20


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ASSOCIATED CONTENT Supporting Information Sample preparation of HRP, avidin, IgG, fetuin, human serum and rat brain; parameters for MS analysis and data analysis; solid-state

13

C NMR spectra;potentiometric titration curves;

photograph of compressive test of the CSMs; FT-IR spectrum and XRD pattern of CSMs; tandem mass spectra of some glycopeptides from HRP, avidin, IgG and fetuin; MALDI mass spectra of HRP digests and the tryptic digest mixtures of HRP and BSA with the Sepharose CL-6B enrichment; detection limit of this method; the MS intensities of six selected glycopeptides from tryptic digests of human IgG (3 µg) after enrichment with different amounts of CSMs; detailed information of glycopeptides obtained from HRP, avidin, IgG, fetuin, human serum and rat brain. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure Captions Figure 1. (a-c) Preparation process of CSMs. (a) The transparent chitosan alkaline solution, (b) the chitosan pre-gel solution in isooctane/Span 80 under stirring in the ice-water bath

and (c)

the final fabricated chitosan microspheres; (d) Proposed schematic representation for the crosslinking reaction of chitosan with epichlorohydrin.

Figure 2. SEM image (a) and size distribution (b) of CSMs. SEM image of the representative surface (c, d) and optical photomicrograph (e) of CSMs.

Figure 3. Nitrogen adsorption-desorption isotherm (a) and BJH pore-size distribution curve (b) of CSMs; the swelling ratios of CSMs in different pH values (c); the compressive stress-strain curve of CSMs (d).

Figure 4. MALDI mass spectra of tryptic digests of HRP. Direct analysis (a), analysis after enrichment with CSMs (b), and analysis of the sampling eluate (c); analysis after enrichment with GA cross-linked CSMs (d). Glycopeptides are marked with ‘Hn’.

Figure 5. MALDI mass spectra of the tryptic digest mixtures of HRP and BSA without (a, c) or with (b, d) the CSMs enrichment. Molar ratios of HRP to BSA are 1: 10 (a, b) and 1: 100 (c, d). Glycopeptides are marked with ‘Hn’.

Figure 6. MALDI mass spectra of tryptic digests of avidin: direct analysis (a), analysis after 24


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enrichment with CSMs (b), and analysis of the deglycosylated peptides enriched by CSMs (c); MALDI mass spectra of tryptic digests of IgG: direct analysis (d), analysis after enrichment with CSMs (e), and analysis of the deglycosylated peptides enriched by CSMs (f). Glycopeptides are marked with ‘An’ or ‘In’.

Figure 7. ESI mass spectra of tryptic digests of fetuin: direct analysis (a), analysis after enrichment with CSMs (b). Glycopeptides are marked with their m/z and charge.

Figure 8. Analysis of the motif composition of identified N-glycosylation sites from rat brain after enrichment with CSMs (a), and analysis of the distribution of single N-glycosylation site and multiple N-glycosylation sites in detected glycoproteins (b).

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Figure 1

26


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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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For ‘TOC’ only,

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High Strength and Hydrophilic Chitosan Microspheres for the

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