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Bioinspired Saccharide-Saccharide Interaction and Smart Polymer for Specific Enrichment of Sialylated Glycopeptides Xiuling Li, Yuting Xiong, Guangyan Qing, Ge Jiang, Xianqin Li, Taolei Sun, and Xinmiao Liang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03104 • Publication Date (Web): 13 May 2016 Downloaded from http://pubs.acs.org on May 17, 2016

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ACS Applied Materials & Interfaces

Bioinspired Saccharide-Saccharide Interaction and Smart Polymer for Specific Enrichment of Sialylated Glycopeptides Xiuling Li, †, § Yuting Xiong, ‡, § Guangyan Qing*, ‡Ge Jiang,† Xianqin Li,† Taolei Sun*‡ and Xinmiao Liang*,† †

Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical

Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, P. R. China. ‡

State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan

University of Technology, 122 Luoshi Road, Wuhan 430070, P. R. China. KEYWORDS: smart polymer, saccharide, glycopeptide, sialic acid, enrichment, hydrogen bond

ABSTRACT: Abnormal sialylation of proteins is highly associated with many major diseases, such as cancers and neurodegenerative diseases. However, this study is challenging owing to the difficulty in enriching trace sialylated glycopeptides (SGs) from highly complex biosamples. The key to solving this problem relies strongly on the design of novel SG receptors to capture the sialic acid (SA) moieties in a specific and tunable manner. Inspired by the saccharide-saccharide interactions in life systems, here we introduce saccharide-based SG receptors into this study.

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Allose (a monosaccharide) displays specific and pH-sensitive binding toward SAs. Integrating allose units into a polyacrylamide chain generates a saccharide-responsive smart copolymer (SRSC). Such design significantly improves the selectivity of SA binding, meanwhile, this binding can be intelligently triggered in a large extent by solution polarity and pH. As a result, SRSC exhibits high-performance enrichment capacity toward SGs, even under 500-fold interference of bovine serum albumins digests, which is notably higher than conventional materials. In real biosamples of HeLa cell lysates, 180 sialylated glycosylation sites (SGSs) have been identified using SRSC. This is apparently superior to those obtained by SA-binding lectins including WGA (18 SGSs) and SNA (22 SGSs). Furthermore, lactose displays good chemoselectivity toward diverse disaccharides, which indicated the good potential of lactose-based material in glycan discrimination. Subsequently, the lactose-based SRSC facilitates the stepwise isolation of O-linked or N-linked SGs with the same peptide sequence but varied glycans by CH3CN/H2O gradients. This study opens up a new avenue for next generation of glycopeptide enrichment materials.

1. INTRODUCTON Protein glycosylation is one of the most common and intricate post-translational modifications in mammals, because of its highly variable carbohydrate compositions, linkages, branching structures, and its intra- and inter-species diversity.1-5 Sialic acids (SAs), a class of 9-carbon backbone monosaccharides, are expressed at the outer ends of glycan chains on vertebrate cell surfaces. These sialylated glycans play fundamental roles in cell–cell and cell–microenvironment interactions.6-9 The increased expression of SAs in glycoproteins has been extensively discovered


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in the sera of patients with different kinds of cancers, such as breast and lung cancers.10-14 However, the profiling of protein sialylation by mass spectrometry (MS) is largely hampered by the obstacle to enrich trace sialylated glycopeptides (SGs, approximately 0.01–0.03% in total amount of peptides) from highly complex bio-samples.15-20 The commonly used glycopeptide (GP) enrichment methods, such as boronate affinity chromatography21-25 and hydrophilic interaction liquid chromatography (HILIC),26-31 usually suffer from low selectivity for GP enrichment and poor anti-interference properties [the most favorable result was not higher than 1:100 for the molar ratio of glycoprotein to bovine serum albumin (BSA)].32-34 TiO2 affinity chromatography has also been used to specifically enrich SGs.35,36 However, the chelating interaction between SGs and TiO2 is too strong and this method lacks adequate tunability, thereby rendering SG elution difficult, which leads to the low recovery and loss of large amounts of SG signals. Destructive hydrazine chemistry based methods show high GP selectivity, the glycan information cannot be obtained owing to the destruction of glycans by violent oxidation reactions.37,38 Lectin affinity chromatography is generally recognized as the most promising enrichment method for GPs owing to the high specific binding of lectins (saccharide-binding proteins) to saccharide moieties.39-41 However, high specificity makes each lectin effective only for a narrow subset of GPs and vast amounts of other information will be lost. For example, sambucus nigra agglutinin II (SNA) specifically binds to α(2-6)-linked SAs, and α(2-3) linked SAs are missed.4244

On the other hand, lectins also have hydrophobic domains, which can combine with

hydrophobic peptides. This causes strong background interference for GP identification by MS. Therefore, an ideal receptor for SG enrichment should be able to selectively bind all SGs, and exhibit good tenability to remove large amount of non-GP interference.45


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Stereospecific saccharide-saccharide interactions at the cell surface (Scheme 1A) emerge as a crucial mechanism for cell adhesion and recognition.17,46-50 Their high specificity is mainly attributed to the multiple hydrogen bond (H-bond) interactions among carbohydrates.51 Inspired by this property, we designed a novel SG receptor based on the saccharide-saccharide interactions. Our study indicated that allose (a monosaccharide) could act as a good SA receptor. Quantum chemistry calculation showed strong binding (six H-bonds, Scheme 1B) between allose and N-acetyl-neuraminic acid (Neu5Ac, a typical SA). We further grafted allose units, using benzene ring as a linker, onto a polyacrylamide (PAM) chain with grafting densities from 3% to 33% (Scheme 1C) and obtained a saccharide-responsive smart copolymer52 (SRSC): PAM-gallose. This design significantly improved the combination with SGs (Scheme 1D), which could be largely modulated by solution pH and polarity [acetonitrile (CH3CN)/H2O ratio]. This brought obvious advantages to optimize the chromatographic conditions in SG enrichment. As a result, SRSC showed excellent SG enrichment capacity. Different binding behaviors of SRSC to SAs and other saccharides further enabled chromatographic separation of SGs and neutral glycopeptides, which would greatly benefit the MS analysis of sialylated glycosylation. More interestingly, the SRSC based on lactose (a disaccharide) allowed the discrimination of different disaccharides, which facilitated the stepwise isolation of SGs with the same peptide sequence but varied glycans.


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Scheme 1. (A) Schematic illustration of complexation between two glycoproteins expressed on the cell membrane, which is driven by multiple H-bond interactions between saccharide moieties17. (B) Optimized interaction model between Neu5Ac and allose, showing six strong Hbonds (green dash lines). This model was obtained by quantum chemistry calculation (Gaussian, density functional theory, at the 6-311G level, solvent parameter: H2O, pH: 4). (C) Chemical structure of PAM-g-allose0.10 grafted copolymer. (D) Possible binding mode of PAM-gallose@SiO2 to SGs with complex glycan structures.

2. EXPERIMENTAL DETAILS 2.1. Materials. The saccharide-responsive smart copolymer PAM-g-allose was constructed by PAM main chains (Sigma-Aldrich Corp.) grafted with 4-Formylphenyl β–D-allopyranoside


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(Tokyo Chemical Industry (TCI) Co., Ltd (Tokyo, Japan)) units. Various monosaccharides were also purchased from (TCI) Co., Ltd. Double distilled water (18.2 MΩ cm, Milli-Q system) was used in the experiments. Other materials and instruments are described in Part S1 in the supporting information (SI). 2.2. Synthesis of monosaccharide-based fluorescent sensors. An automated microwave tube (30 mL) was charged with monosaccharide (i.e. D-allose, D-glucose, D-galactose, D-mannose or L-fucose, 0.96 g, 6.0 mmol), ammonium carbonate ((NH4)2CO3, 4.80 g, 50 mmol) and anhydrous DMSO 15 mL. The tube was sealed and placed in an automated microwave synthesizer (Biotage Initiator 8 EX), the reaction was proceeded at 40 °C in 10 watts power for 4 hours. Then the reaction mixture was freeze-dried to remove the excess of ammonia and DMSO to obtain β-glycosyl amine. Then β-glycosyl amine (0.18 g, 1.0 mmol), fluorescein isothiocyanate (FITC, 0.39 g, 1.0 mmol), and 5.0 mL anhydrous DMF were added to a 25 mL round-bottom flask. The reaction mixture was stirred at room temperature overnight. After that, the solvent was removed under reduced pressure. The crude product was purified on a Shimadzu UFLC 20A purity system with a C18 reversed-phase analytic chromatographic column [250 mm×10 mm inner diameter (I.D.), Boston Analytics, Corp. China] to afford FITC-labeled Dallose, which was confirmed by ESI Q-TOF MS (m/z calcd.: 568.12, found: 569.12 [M+H]+). Similar procedure was adopted to prepare FITC-labeled D-glucose (m/z calcd.: 568.12, found: 569.12 [M+H]+), FITC-labeled D-galactose (m/z calcd.: 568.12, found: 569.12 [M+H]+), FITClabeled D-mannose (m/z calcd.: 568.12, found: 569.12 [M+H]+) and FITC-labeled L-fucose (m/z calcd.: 552.12, found: 553.12 [M+H]+). 2.3. Synthesis of PAM-g-allose with different grafting densities (Scheme S1 in SI). A 50 mL round-bottom flask was added a certain amount of 4-formylphenyl β-D-allopyranoside 1.0 g,


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polyacrylamide (PAM) 2.5 g, sodium carbonate 0.5 g, 10 mL water and 15 mL methanol. After stirring for 48 hours at 60 °C, the crude product was obtained, and purified by dialysis in a water /methanol mixture solvent (50/50, v/v) for 3 days, using a piece of dialysis membrane (2 cm×10 cm, molecular weight Cut-Off 3000). White powder of PAM-g-allose0.10 was obtained after freeze-drying. The final grafting densities were determined by calculating the ratios of integral areas of the characteristic peaks of D-allose to those of PAM in the 1H NMR spectra (Figure S1 in SI). 1H NMR (500 MHz, deuterated water (D2O)): δ (ppm): 1.26-1.69 (m, 20H, C-CH2), 1.922.24 (m, 10H, C-CH), 3.39-3.96 (m, 5H, CH-OH and CH2-OH), 4.21 (t, J=3.0 Hz, 1H, CH-OH), 5.43 (d, J=8.1 Hz, 1H, O-CH-O), 7.21 (d, J=8.7 Hz, 2H, Ph-H), 7.89 (d, J=7.1 Hz, 2H, Ph-H), 9.75 (s, 1H, CH=N). IR (cm–1): 3346, 3202, 2927, 1658, 1606, 1579, 1510, 1452, 1429, 1399, 1350, 1322, 1257, 1172, 1115, 1102, 1072, 1036, 912, 838, 821, 804, 701, 611. Elemental analysis for PAM-g-allose0.10 found: C, 52.75; H, 14.53; N, 25.96. Using the same method, PAM-g-allose with different grafting densities (i.e., PAM-g-allose0.03, PAM-g-allose0.13, PAM-gallose0.16, PAM-g-allose0.19, and PAM-g-allose0.33) could be obtained by changing the amount of 4-formylphenyl β-D-allopyranoside. 2.4. Synthesis of PAM-g-allosex@SiO2 enrichment materials. (a) Silica gels (5.0 g, average particle size: 5 µm and average pore diameter: 300 Å) were suspended in 25 mL of hydrochloric acid (HCl, 0.1 mol·L‒1 (M)) for 48 hours at ambient temperature to generate sufficient hydroxyl groups on the silica surface. The hydroxyl activated silica gels were separated by centrifugation at 7000 rpm for 5 min. Then, the silica gels were washed three times with ultrapure water and then ethanol by repetitive dispersion/precipitation cycles, and then the silica gels were dried under vacuum.


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(b) 3-triethoxysilylpropyl isothiocyanate (3.0 mL) was dissolved in anhydrous toluene (40 mL), and the aforementioned silica gels (5.0 g) were added. The mixture was stirred and refluxed for 6 hours. The product was separated by centrifugation at 7000 rpm for 5 min. Then, the isothiocyanate-modified silica gels (denoted as NCS@SiO2) were washed three times with toluene and then ethanol by repetitive dispersion/precipitation cycles to remove the unreacted materials, and then the silica gels were dried under vacuum. (c) 0.20 g PAM-g-allosex (x=0.03, 0.10, 0.13, 0.16, 0.19 or 0.33, respectively) was allowed to react with 0.50 g NCS@SiO2 in ultrapure water (25 mL) for 2 days. The PAM-g-allosex modified silica gels (denoted as PAM-g-allosex@SiO2) were separated by centrifugation at 7000 rpm for 5 min. Then, PAM-g-allosex@SiO2 was washed three times with ultrapure water and then ethanol by repetitive dispersion/precipitation cycles to remove the unreacted materials, and then the silica gels were dried under vacuum. Pure PAM@SiO2 was obtained by using the same method. 2.5. Fluorescent titration experiment. To investigate the binding properties (Ka) of D-allose for diverse monosaccharides (i.e. D-glucose, D-galactose, GlcNAc, GalNAc, D-mannose, Lfucose, D-lyxose, D-xylose and Neu5Ac) at pH 7.4 and pH 4.0, a series of fluorescent titration experiments were conducted. Host FITC-labeled D-allose was prepared as stock solution in Trisbuffer solution (10 mM, pH 7.4) and formate-buffer solution (10 mM, pH 4.0) for 5.0×10‒6 mol·L‒1, respectively. Guest monosaccharides were prepared to 1.75×10‒3 and 1.75×10‒2 mol·L‒1 of stock solutions in H2O. The work solutions were prepared by adding different volumes of guest solutions to a series of test tubes, and then same amount of stock solution of host FITClabeled D-allose was added into each test tube, followed by dilution to 3.00 mL by Tris-buffer solution or formate-buffer solution. After being shaken for 1 minute, the work solutions were


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measured immediately at 20 oC using a Perkin-Elmer LS-55 spectrometry. Association constant (Ka) were obtained according to intensity changes at the maximum emission peak. Similar method was adopted to investigate the binding capacities of five host FITC-labeled monosaccharides with Neu5Ac, D-glucose, D-galactose, or GlcNAc in formate-buffer solution (10 mM, pH 4.0) or Tris-buffer solution (10 mM, pH 7.4), respectively. 2.6. Adsorption Monitored by Quartz Crystal Microbalance with Dissipation (QCM-D). An Au-coated QCM resonator was firstly cleaned by a fresh mixture solution of distilled water, ammonium hydroxide and H2O2 (v/v/v, 5/1/1) for 10 minutes at 70 °C. After being rinsed by double distilled water sufficiently and dried under a flow of nitrogen gas, the resonator was immersed

in

10

mL

water

containing

(((2-mercaptoethyl)imino)methyl)phenyl

β-D-

allopyranoside (0.10 g, the preparation process see Scheme S2 in SI) for 24 hours, allowing the self-assembly of a monolayer D-allose on the QCM-D resonator surface via an Au-S bonding reaction. The allose-modified resonator was washed with distilled water for three times, then dried by a flow of nitrogen gas and was put into a flow-cell for frequency measurement, in which the cell temperature was maintained at 25 °C. After stabilization of fundamental resonance frequency with pure water and pH buffer solution (10 mmol·L‒1), monosaccharide buffer solution (25 mmol·L‒1, i.e. Neu5Ac, GlcNAc or D-galactose) was pumped into flow-cell by a peristaltic pump at a constant speed of 0.100 ml·min‒1, respectively. The frequency change was recorded by Q-Sense software and analyzed by Q-Tools. 2.7. 1H NMR titration experiments. In order to investigate the binding details between Dallose and other monosaccharides (i.e. Neu5Ac, GlcNAc or D-galactose), 1H NMR titration experiments were conducted. D2O was chosen as the solvent because all these monosaccharides


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are well soluble in it. D-allose was allowed to mix with Neu5Ac at a molar ratio of 1:1, concentrations: 2 mmol. After equilibration for 4 hours, the chemical shifts of C-H protons in monosaccharides were recorded by NMR spectrometer. Similar 1H NMR titration experiments were also conducted in the investigation of the complexation between D-allose and GlcNAc or D-galactose at a molar ratio of 1:1, as shown in Figure S7 and S8 in SI. 2.8. Contact angle experiment. The static contact angle (CA) measurement was conducted using the sessile drop method on a DataPhysics OCA35 goniometer with software SCA20 at ambient atmosphere and a constant temperature of 25 °C. The solution of CH3CN/H2O (v/v=4:1) was prepared precisely. The allose monolayer, Sepharose, PAM- or PAM-g-allosex-modified silicon substrates were immersed in the distilled water or 80% CH3CN/H2O for 15 minutes, respectively, and then these surfaces were dried under a flow of nitrogen gas. After that, the contact angle was measured immediately, each experiment was performed in quadruplicate in order to obtain an average CA value. 2.9. Enrichment of glycopeptides with Enrichment of glycopeptides with allose@SiO2, PAM@SiO2, PAM-g-allose0.10@SiO2. Glycopeptide enrichment was performed in a microscale solid phase extraction (SPE) mode. Eppendorf GELoader tip was packed with 1 mg PAMg-allose0.10@SiO2 materials after being slurried with acetonitrile (ACN). The tips were conditioned and equilibrated with 20 µL 50% ACN/20 mM ammonium formate (NH4FA) (pH=3.8) and 80% ACN/20 mM NH4FA (pH=3.8). After loading tryptic digests redissolved with 80% ACN/20 mM NH4FA, the tips were rinsed twice with 20 µL 75% ACN/20 mM NH4FA and 75% ACN/0.1% formic acid (FA), subsequently. The trapped glycopeptides were eluted with 30 µL 50% ACN/5 mM NH4HCO3 (pH=8.6). Similar optimized protocol was adopted when


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allose@SiO2 (Figure S15 in SI) and PAM@SiO2 (Figure S30c in SI) were compared to enrich glycopeptides. 2.10. Stepwise separation of SGs with variable glycans. Stepwise elution of SGs was carried out with PAM-g-lactose0.06@SiO2. Briefly, 1.0 mg PAM-g-lactose0.06@SiO2 were loaded into the GE-Loader tips and conditioned with 20 µL 50% CH3CN/0.1% TFA. After equilibration with 20 µL 80% CH3CN/20 mM NH4FA (pH=3.8), tryptic digests were loaded into the tips. Then the micro-column was sequentially washed with 20 mM NH4FA containing 75% CH3CN, 70% CH3CN, 65% CH3CN, 60% CH3CN and 55% CH3CN, respectively. Each elute was dried with Speed Vac and desalted with C18 before MS analysis. Other experimental details see Part S2 and S3 in SI.

3. RESULTS AND DISCUSSIONS 3.1. Allose-based SA Receptor Our first step was to develop a receptor capable of discriminating Neu5Ac from other neutral saccharides. The binding capacities (i.e. association constants: Ka) of allose for various monosaccharides were obtained by fluorescent titration experiment,53,54 which is a typical and rapid method for measuring affinity constant between host and guest molecules in supramolecular chemistry.55 As shown in Figure 1A, at the physiological pH of 7.4, FITClabelled allose exhibited variable binding properties for different monosaccharides, and the Ka value for galactose (1×105 L·mol–1) was the highest. By contrast, Ka for Neu5Ac was only 597 L·mol–1. However, this value substantially increased to 5.2×105 L·mol–1 (this large Ka could also be observed for other carbohydrate-receptors56,57) when the solution pH changed to 4.0, whereas


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no evident changes in Ka were observed for other neutral monosaccharides (Figure 1B). This phenomenon was further proved by the detailed pH scan of Ka for Neu5Ac, N-acetylglucosamine (GlcNAc) and galactose. As shown in Figure 1C, Ka for Neu5Ac reached the peak value at pH 4.0, which was about 871 times larger than that at pH 7.4. By contrast, for GlcNAc and galactose, the Ka values did not change significantly with the change of pH. Besides, we also attempted to validate the Ka values through isothermal titration calorimetry experiment (usually suitable for calculation of Ka between biomacromolecules) or 1H NMR titration experiment (Figure S9 and S10 in SI), but the corresponding changes in heat release or chemical shifts were not obvious enough to obtain reliable Ka data between allose and Neu5Ac.


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Figure 1. pH-mediated binding behavior of allose toward Neu5Ac. (A, B) Association constants (Ka) of allose interacted with various monosaccharides in buffer solutions at pH 7.4 (A) or pH 4.0 (B), at 20 oC, obtained by fluorescent titration experiments. (C) Effect of solution pH on Ka values of allose with Neu5Ac, GlcNAc, or galactose, respectively. (D) pH-dependent frequency changes (∆F) upon adsorption of Neu5Ac, GlcNAc, or galactose on allose monolayer surface, obtained by a quartz crystal microbalance (QCM) adsorption experiment. (E–G) Partial hydrogen nuclear magnetic resonance (1H NMR) spectra of Neu5Ac (E), allose (F) and a mixture of Neu5Ac with equimolar allose (G) in water (D2O) at pH 3.8 and at 20 oC. Upfield shifts of the CH protons of allose or SA are indicated by black or red dotted lines, respectively. (H, I) Orthogonal studies to screen out the optimal Neu5Ac receptor. Ka values of Neu5Ac and glucose with different monosaccharide hosts in aqueous solutions at pH 4.0 (black column) and pH 7.4 (red column) are shown in (H) and (I), respectively. Correspondingly, the adsorption experiments of Neu5Ac, GlcNAc and galactose on allose monolayers using quartz crystal microbalance (QCM) (Figure 1D) exhibited the same tendency.57-59 For Neu5Ac, the maximum adsorption (∆F: 16 Hz, corresponding to an adsorption quantity of 94 ng·cm–2) also occurred at pH 4.0. Those for GlcNAc and galactose did not show any responsiveness to pH, which were at the same level as those of Nue5Ac at low and high ends of pH (2.0 and 9.0) —all were much smaller than that of Neu5Ac at pH 4.0. Hydrogen nuclear magnetic resonance (1H NMR) spectra (Figure 1E-G) show the details on binding in an acidic aqueous solution.60 Almost all C-H proton of allose and some protons of SA exhibited clear upfield shifts, revealing intensive H-bond interactions between Neu5Ac and allose. Under the same condition, no obvious change in the 1H NMR spectra was observed for the complexation


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between allose and galactose or GlcNAc [Figure S7 and S8 in SI]. These results clearly showed a pH-dependent manner for allose/Neu5Ac binding. The pH-dependence of binding behavior was also investigated using the other four monosaccharide-based receptors, in an attempt to identify the most favorable Neu5Ac receptor. As shown in Figure 1H, the four tested saccharide-based receptors, namely allose, galactose, mannose and fucose, but not glucose, displayed considerably stronger binding capacities for Neu5Ac at pH 4.0 than at pH 7.4. Among them, allose showed the largest difference in Ka values with a Ka (pH 4.0)/Ka (pH 7.4) ratio of 850, which was considerably larger than those observed for glucose (0.9), galactose (40.8), mannose (28.4) and fucose (57.2). Under the same condition, when neutral galactose, glucose and GlcNAc (Figure S5 in SI) were used as guests, no significant difference was observed in Ka values between different monosaccharide hosts and guests at pH 4.0 and pH 7.4, as typically shown in Figure 1I with galactose as the guest. These data clearly demonstrated that allose could discriminate Neu5Ac from other neutral saccharides more effectively utilizing its pH-mediated binding abilities. In addition, allose exhibited considerably stronger binding with Neu5Ac than with other acids, such as gluconic acid, tartaric acid and ascorbic acid (Figure S6 in SI). This implied that the highly specific complexation between allose and Neu5Ac was not attributed to the acidity of Neu5Ac, but rather to its unique structure. 3.2. Allose-Based Smart Polymers for Glycopeptide Binding Allose was further immobilized onto silica gels to capture SGs from tryptic digests of bovine fetuin (a sialylated glycoprotein) with different interference levels of BSA. Unexpectedly, the allose-monolayer coated material (a kind of hydrophilic material with intrinsic contact angle


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(CA) of about 22o) exhibited strong affinities to both GPs and hydrophilic non-GPs, leading to a poor selectivity, which could not resist even 5-fold interference of BSA (see Figure S15 in SI). The retention of peptides decreased as the water content of CH3CN/H2O mixture increased, indicating that this material was a typical HILIC material (Figure S16 in SI). According to the classical partitioning retention mechanism in HILIC,61 the water-rich layer near the allose monolayer (the light blue layer in Figure 2A and 2B) considerably strengthened the hydrophilic interactions of polar solutes (e.g., non-GPs, neutral GP and salts); however, it also obstructed direct contact between allose and SAs at the distal ends of SG. Neu5Ac must compete with other hydrophilic solutes and repel the water cluster, to approach the matrix–binding domains, which is a common challenge in saccharide recognition in the aqueous phase.62-64 This mainly weakened the affinity of the material for SGs, resulting in poor selectivity.


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Figure 2. Illustration of the partitioning retention mechanism in hydrophilic interaction liquid chromatography (HILIC) with allose monolayer (A) or Sepharose (B) material. Possible polymer conformation and saccharide-binding models of PAM-g-allose with a grafting ratio of 0.33 (C) and 0.10 (D), respectively. H-bonds are indicated by blue dashed lines. Poor enrichment selectivity was also observed with the commercial Sepharose HILIC material, which is a cross-linked polysaccharide widely used in glycoproteomics. We assume that the presence of a water-rich layer may not be beneficial in improving the selectivity of the material. Furthermore, because of the extensive intermolecular H-bond interactions among saccharide monomers, the polysaccharide chains tend to wrap around each other to form a contracted and rigid polymeric micelle (Figure 2B). Consequently, only small amounts of hydroxyl groups remain exposed and can bind to exterior solution. This reduced the performance of polysaccharide-based materials in GP enrichments (Figure S17 in SI). The aforementioned analysis indicates that the surface wettability and the grafting ratio of allose are two critical factors in designing materials.


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Figure 3. (A) Profiles of water droplets on different material surfaces treated with pure water (top panel) or a CH3CN/H2O mixture (bottom panel). (B) Determination of the optimal grafting ratio of PAM-g-allose for binding with sialylated glycopeptides (SGs). Adsorption capacities of allose monolayers, PAM-modified silica gels, Sepharose and PAM-g-allosex@SiO2, toward SGs in fetuin tryptic digests. To overcome the problem caused by the water-rich layer, we grafted allose units onto PAM chains and developed a serial of PAM-g-allosex materials with grafting ratios (x) from 3% to 33%, which were further immobilized onto silica gels (denoted as PAM-g-allosex@SiO2). CA measurements indicated that these materials were relative hydrophobic (Figure 3A), possibly


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because of the coil state of the polymer chains (Figure 2C).65 The CA values increased gradually from 74o to 82o with the increase of grafting ratio from 3% to 33% (Figure 3A). We also observed significant hydrophobicity increase when we treated the film with a CH3CN/H2O mixture (volume ratio: 4:1) (Figure 3A bottom), which was consistent with the practical GP enrichment condition that would be involved in the following experiment. The hydrophobicity of the copolymer surface could suppress the non-specific hydrophilic interactions with polar solutes. These PAM-g-allosex@SiO2 materials displayed much higher adsorption capacities (Figure 3B) for SGs of fetuin tryptic digests (Figure S14 in SI) than Sepharose (~10 mg·g–1) and the allose-monolayer modified silica gels (allose@SiO2, ~30 mg·g–1), and the highest capacity (220 mg·g–1) turned up at an intermediate allose grafting ratio (x=0.10). In a control experiment, pure PAM@SiO2 showed a rather low capacity (10 mg·g–1), indicating that PAM only acted as a polymeric framework and allose units provided the main binding sites for SGs. This was also confirmed by the 1H NMR titration experiments (Figure S12 in SI). Nearly all C-H protons of Neu5Ac displayed obvious upfield shifts when complexed with PAM-g-allose0.10. Meanwhile, the C-H protons of allose appending in the polymeric chain also exhibited upfield shifts. By comparison, no evident changes in chemical shifts of the aromatic protons in benzene rings were observed, which further demonstrated the bridging role of benzene. In addition to grafting ratio of allose, polymer conformation is another major factor determining the performance of PAM-g-allosex in SG adsorption. For the medium or low allose ratio (e.g., 10% or lower, Figure 2D), copolymer chains preferred to exhibit a “relaxed” state and most allose units remained exposed, which provided sufficient binding sites for SGs. Nevertheless, for a high allose ratio (e.g., 33%, Figure 2C), intensive H-bond interactions between allose and neighboring allose units or PAM amides resulted in the contraction and


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aggregation of copolymer chains, which largely reduced the number of allose units exposed on the surface. This would strongly suppress the adsorption of SGs. The presumption was confirmed by the static light scattering experiment (Figure S13 in SI), which clearly showed the aggregation state of PAM-g-allose0.33 and the relaxed state of PAM-g-allose0.10 in an aqueous solution. 3.3. Selective Enrichment of SGs from Complex Biosamples We then compared the chromatographic retention behaviors of neutral GPs and SGs on PAMg-allose0.10@SiO2 (Figure S18 in SI). Since allose had significantly higher affinity to Neu5Ac than to other neutral saccharides at pH 4.0, which became rather weak at pH 7.4, we successfully realized stepwise separation of GPs and SGs from digests of horseradish peroxidase (HP, a neutral glycoprotein) and fetuin mixture by controlling the pH of loading and elution buffers (Scheme S3 in SI). GPs from HP were detected in the flow-through and fraction eluted with 70% CH3CN/50 mmol·L–1 ammonium formate (NH4FA, pH=3.8), whereas SGs from fetuin were detected only in the fraction with 50% CH3CN/5 mmol·L–1 ammonium bicarbonate (NH4HCO3, pH 8.6).


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Figure 4. Glycopeptide (GP) enrichment by PAM-g-allose0.10. (A) pH-mediated enrichment strategy based on a micro-SPE mode. (B, C) Mass spectra of GPs enriched with PAM-gallose0.10@SiO2 from the tryptic digests of fetuin and BSA at molar ratios of 1:200 (B) and 1:500 (C). Non–GPs are labeled with their m/z values and GPs are marked with red stars (B) or their glycan structures (C): blue square: GlcNAc; green circle: mannose; yellow circle: galactose; purple diamond: Neu5Ac.


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We then evaluated the enrichment selectivity of PAM-g-allose0.10@SiO2 for SGs in a microscale solid phase extraction (SPE) mode (Figure 4A), by using tryptic digests of bovine fetuin with different interference levels (e.g., 1:200 and 1:500 in molar ratios) of BSA (a typical nonglycosylated protein) interference. Before enrichment, all peptide signals in the mass spectrum (Figure S19 in SI) were originated from non-GPs. Figure 4B showed the mass spectrum of a fetuin sample mixed with 200-fold BSA interference after enrichment by using a pH-mediated rinsing protocol. The majority of non-GPs were efficiently removed, resulting in 36 readily characterized GP signals. For a higher interference level (500-fold BSA), the GP selectivity remained quite well and 15 GP signals were identified (Figure 4C). Tandem MS analysis indicated that all these signals were SGs (Figures S21–S29 in SI). Lectins have been generally recognized as the best receptors capable of binding saccharides specifically. And lectin affinity chromatography is the preferred purification method in glycoproteome. For comparison, two SA-specific lectins—wheat germ agglutinin (WGA) and SNA had poor enrichment selectivity for GPs from the mixture of fetuin and BSA at the molar ratio of 1:10 (Figures S31 and S32 in SI). With the same peptide mixture, other commercial materials (i.e. Sepharose and ZIC-HILIC) also exhibited quite low enrichment selectivity (Figures S18 and S34 in SI). These results further demonstrated that PAM-g-allose0.10@SiO2 had higher selectivity for SGs than other materials including Sepharose and lectins. Furthermore, PAM-g-allose0.10@SiO2 was used to enrich GPs from the tryptic digests of a HeLa S3 cell lysate. On average, more than 200 SGs with 180 unique sialylated glycosylation sites were successfully characterized from 50 µg protein samples in two replicated experiments (Figure 5A). The detailed information of the identified glycosylation sites is shown in Table S1 in SI. By contrast, when SA-specific binding proteins, namely WGA and SNA, were tested with


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the same amount of protein samples (experimental details see SI), the numbers of identified sialylated glycosylation sites were 19 and 28, respectively, all of which were substantially less than the numbers identified by using our material. Gene ontology analysis indicated that 86% of matched glycoproteins were annotated as binding proteins, these are known to be involved in the response to stimuli and wounding (Figure 5B).66 These data exemplify the excellent potential of our material for use in glycoproteomic analysis.


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Figure 5. (A) Comparison of the sialylated glycosylation sites identified from a HeLa S3 cell lysate with PAM-g-allose0.10@SiO2, WGA and SNA (two typical SA-specific binding proteins). (B) Molecular functions of glycoproteins identified by PAM-g-allose0.10@SiO2 material, determined through gene ontology analysis. 3.4. Stepwise Separation of Different SGs with Variable Glycans. Our material design concept can be easily expanded from monosaccharide to disaccharide. Lactose, the most abundant oligosaccharides in human milk, also exhibited strong complexation with Neu5Ac and the Ka value was 5.5×105 L·mol–1 (obtained from the fluorescent titration experiment) in formate-buffer solution at pH 4.0. Thus, lactose units were grafted onto PAM chains at a grafting ratio of 6%, and PAM-g-lactose0.06 was then bound onto silica gels (denoted as PAM-g-lactose@SiO2). PAM-g-lactose@SiO2 was packed into GELoader tips and the resulting SPE microcolumn was used to evaluate the retention behavior of different monosaccharides (i.e. mannose, galactose, GlcNAc and Neu5Ac). The results indicated that PAM-g-lactose@SiO2 had much stronger retention toward Neu5Ac than other neutral monosaccharides (Figure S32 in SI). Further application of PAM-g-lactose@SiO2 to enrichment of SGs resulted in 15 SG signals from the tryptic digest of fetuin mixed with 200-fold BSA interference (Figure S33B in SI). More interestingly, Ka measurement (Figure S34 in SI) revealed that lactose could discriminate five disaccharides (i.e. sucrose, maltose, lactose, cellobiose and trehalose), which differ in their monosaccharide compositions or linkage modes. This indicated the good potential of our materials in glycan discrimination. Furthermore, PAM-g-lactose@SiO2 was applied to discriminate SGs with the same peptide sequences but different glycan structures (Table 1).


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When fetuin GPs were successively eluted with a reduced content of acetonitrile from the material, GPs differing in only one SA could be distinctly separated into different fractions, indicating the high separation efficiency of material for SGs. This feature can considerably reduce the complexity of GP sample and help biologists acquire more accurate information about glycans.15, 67


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Table 1. Stepwise elution of O-linked or N-linked SGs from lactose-based polymer by solvent polarity. Peptide sequences: Pep. 1: VTCTLFQPVIPQPQPDGAEAEAPS(271)AVPDAAGPTP SAAGPPVASVVVGPSVVAVPLPLHR; Pep. 2: VVHAVEVALATFNAESN(176)GSYLQLV EISR. Glycan structure: blue square: GlcNAc; green circle: mannose; yellow circle: galactose; purple diamond: Neu5Ac.√: minor portion of SGs; √√: major portion of SGs

4. CONCLUSIONS In conclusion, taking advantages of the H-bond interactions between allose and Neu5Ac, pH– mediated, intelligent binding behavior, and a favorable polymer conformation,68 the saccharideresponsive polymer material exhibited high specificity toward SGs, and could capture trace SGs from both model fetuin/BSA mixture and real HeLa S3 cell lysate. Good glycan discrimination capacity of our materials further realized the stepwise elution of SGs with slight difference in their glycan structures. These features well satisfy the high demand of large-scale glycoproteome analysis, and will facilitate biologists discovering more sialylated glycosites closely related to cancers or Alzheimer's disease.69 On the other hand, selective recognition of saccharides in aqueous solution is still a huge challenge in chemistry.70 As a bio-inspired driving force employed in this study, saccharide-saccharide stereoselective H-bond interaction points out a new avenue for designing artificial saccharide receptor with high specificity, just as Lectins.ASSOCIATED CONTENT Supporting Information. Materials and instruments, synthesis and preparation procedures, experimental details, characterization data and figures, mass spectra for the enrichment of sialylated glycopeptides with PAM-g-allose@SiO2 and reference materials. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author *[email protected]; [email protected]; [email protected]. Author Contributions § Li, X. and Xiong, Y contribute equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank the National High Technology Research and Development Program of China (2012AA020203), the Major State Basic Research Development Program of China (2013CB933002), the National Natural Science Foundation of China (21135005, 21475129, 21275114, 51473131, and 51173142), China National Funds for Distinguished Young Scientists (51325302) for funding support. G. Qing acknowledges Hubei Provincial Department of Education for financial assistance through the “Chutian Scholar” program and Hubei Provincial Natural Science Foundation of China (2014CFA039). REFERENCES [1]

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