Precision Imprinting of Glycopeptides for Facile Preparation of Glycan

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Precision Imprinting of Glycopeptides for Facile Preparation of Glycan-Specific Artificial Antibodies Zijun Bie, Rongrong Xing, Xinpei He, Yanyan Ma, Yang Chen, and Zhen Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01903 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 24, 2018

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Analytical Chemistry

Precision Imprinting of Glycopeptides for Facile Preparation of GlycanSpecific Artificial Antibodies Zijun Bie, Rongrong Xing, Xinpei He, Yanyan Ma, Yang Chen, and Zhen Liu*

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China

* Corresponding author: [email protected]

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Antibodies specific to glycans are essential in many areas for many important fields, including disease diagnostics, therapeutics and fundamental researches. However, due to their low immunogenicity and poor availability, glycans pose serious challenges to antibody development. Although molecular imprinting has developed into important methodology for creating antibody mimics with low cost and better stability, glycan-specific molecularly imprinted polymers (MIPs) still remain rather rare. Herein, we report a new strategy, precision imprinting with alternative templates, for the facile preparation of glycan-specific MIPs. Glycopeptides with desirable peptide length immobilized on a boronate affinity substrate were first prepared as alternative templates through in situ dual enzymatic digestion. A thinlayer was then produced to cover the glycans to an appropriate thickness through precision imprinting. With glycoproteins containing only N-glycans as well as both N- and O-glycans as glycan source, this approach was proved to be widely applicable and efficient. The strategy is particularly significant for the recognition of O-glycans, because enzymes that can release O-glycans from O-linked glycoproteins are lacking. The MIPs exhibited excellent glycan specificity. Specific extraction of glycopeptides and glycoproteins containing certain glycans from complex samples was demonstrated. This strategy opened a new avenue for the facile preparation of glycan-specific MIPs, facilitating glycan-related applications and research.

Keywords: Glycoproteins, Glycan, Precision imprinting, Artificial antibodies, Boronate affinity

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As

a widely existing post-translational modification, glycosylation plays essential roles in

numerous biological processes such as protein folding, protein trafficking, cell-cell adhesion and cell signaling. Aberrant glycosylation can contribute to a variety of diseases such as cancer and congenital disorders of glycosylation, thus glycans are valuable targets for disease diagnosis and therapy. Glycan-specific antibodies are essential in many fields, including disease diagnostics, therapeutics and fundamental researches. However, due to their low immunogenicity and poor availability, glycans have been known to pose serious challenges to antibody development. 1 The lack of glycan-specific antibodies has been a major barrier for the advancement of related studies.2 Molecularly imprinted polymers (MIPs),3-8 also called artificial antibodies or plastic antibodies, are material mimics of antibodies synthesized through polymerization in the presence of a template. Due to their advantages over antibodies, including easier preparation, low cost and better stability, MIPs have found wide applications in various important fields such as catalysis4-7, immunoassays,8,14 sensing,9-17 and separation.18,19 Recently, the combination of boronate affinity20,21 and molecular imprinting has advanced the preparation of MIPs specific to glycoproteins and saccharides.22-29 Because of their excellent binding properties, the prepared MIPs have enabled promising applications, including disease diagnosis,22,30,31 cancer cell/tissue imaging,25,26 single cell analysis,32 and cancer targeted therapy.33 Particularly, the boronate affinity controllable oriented surface imprinting (BACOSI), has allowed for easy and efficient preparation of MIPs specific to glycoproteins, glycans and monosaccharides.24-27 However, this approach requires pure target compound as the template. In fact, glycans are rather difficult to chemically synthesize, and their preparation from naturally occurring species such as glycoproteins is often 3



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difficult because of poor availability of suitable enzymes, tedious purification procedure and low yield. Therefore, new approaches allowing efficient preparation of glycan-specific MIPs are of significant importance. Templating strategy is an essential aspect in molecular imprinting. Regular imprinting methods14-19,22,23 mainly rely on the use of exact target compound as the template. This strategy is simple, but suffers from difficulty when the template compound is poorly available or unstable within the prepolymer solution. Fragment24,34,35 and epitope10,11 imprinting is an innovative strategy, in which a characteristic fragment of the target compound is used as the template. This strategy makes the selection of template flexible, enabling imprinting of rare, unavailable and labile target compounds. Besides, the use of target analogs36-38 or dummy templates39,40,41 is another novel strategy, particularly useful for the imprinting of small compounds. On the other hand, the controllability of imprinting process is another critical aspect. While the imprinting thinlayer is uncontrollable or uncontrolled in conventional methods,

14-19,22

recent advanced methods12,27

unveiled that excellent binding properties can be achieved through controlling the thickness of the imprinting thinlayer. However, to the best of our knowledge, precision imprinting with elaborate templating to fulfil challenging tasks has not been reported yet so far. Herein we propose a new strategy, called precision imprinting with alternative templates, for the facile preparation of glycan-specific MIPs. In this strategy, glycopeptides with desirable peptide length are prepared using a new approach called in situ dual enzymatic digestion (ISDED) and used as alternative templates. To achieve glycan specificity, a precision imprinting approach was 4


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developed on the basis of our previous tetraethyl orthosilicate (TEOS)-based BACOSI,24 which allows for the imprinting of only the glycan moiety of the alternative template while excluding the peptide chain from the imprinting. The principle and procedure of the ISDED-BACOSI approach are illustrated in Figure 1. First, an intact glycoprotein containing the desired glycan(s) is immobilized onto a boronic acid-functionalized substrate through boronate affinity binding. Then, ISDED is performed to produce glycan-linked short peptide. After that, an imprinting thinlayer is created to cover the glycan to an appropriate thickness through precision imprinting using the TEOS-based BACOSI protocol. Finally, after the template glycopeptide is removed with an acidic solution, an MIP specific to the glycan is obtained, which allows for rebinding with species containing the glycan, such as the intact glycoprotein and glycopeptides digested from the glycoprotein. With glycoproteins containing only N-glycans as well as both N- and O-glycans as glycan source, this approach was proved to be widely applicable and highly efficient. In situ enzymatic digestion has been employed to prepare glycan templates24 and peptide templates,42 which can avoid tedious chemical synthesis and purification of templates. In addition to this merit, ISDED holds significant value for the preparation of glycan templates particularly O-glycans, since it allows for effective preparation of both N-glycans and O-glycans. As a contrast, N-glycans with structure of 1→3 fucose cannot be digested by PNGase F while no suitable enzymes are available for the cleavage of O-glycans. On the other hand, the ISDED-BACOSI approach inherits the merits of the BACOSI strategy, including high imprinting efficiency and excellent binding properties. Thus, this strategy opened a new avenue for the facile preparation of glycan-specific MIPs.

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EXPERIMENTAL SECTION Reagents and Materials. Ribonuclease A (RNase A), ribonuclease B (RNase B), horseradish peroxidase (HRP), transferrin (TRF), bovine serum albumin (BSA), alpha 2-Heremans-Schmid glycoprotein (AHSG), adenosine, deoxyadenosine, trypsin, pronase E, siapinic acid (SA), 2,4difluoro-3-formyl-phenylboronic

acid

(DFFPBA),

tetraethyl

orthosilicate

(TEOS),

3-

aminopropyltriethoxysilane (APTES) and α-cyano-4-hydroxycinnamic acid (CHCA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium cyanoborohydride was from J&K scientific (Shanghai, China). Human serum was purchased from Shuangliu Zhenglong Chemical and Biological Research Laboratory (Sichuan, China). Ferric trichloride hexahydrate, 1,6hexanediamine, anhydrous sodium acetate, glycol, ammonium bicarbonate, sodium chloride, acetic acid, trifluoroacetic acid (TFA), hydrochloric acid (HCl), ammonium hydroxide (28%), anhydrous methanol and anhydrous ethanol were purchased from Nanjing Reagent Company (Nanjing, China). All other reagents used were of analytical grade or higher. Water used in all the experiments was purified by a Milli-Q Advantage A10 water purification system (Millipore, Milford, MA, USA). Instruments. Transmission electron microscopy (TEM) characterization was performed on a JEM1010 system (JEOL, Tokyo, Japan). Vibrating sample magnetometry (VSM) analysis was performed on an MPMS SQUID VSM system (Quantum Design, USA). Fourier transform infrared 6


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(FT-IR) spectrometry was carried out on a Nicolet 6700 FT-IR spectrometer (Thermo Fisher, MA, USA). Ultraviolent (UV) spectral analysis was performed with a NanoDrop 2000/2000C spectrophotometer (Thermo Fisher, MA, USA). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses were carried out on a 4800 plus MALDI-TOF/TOF Analyzer (Applied Biosystems, Framingham, MA, USA) with a pulsed nitrogen laser operated at 337 nm. The laser energy was adjusted to slightly above the threshold to obtain good resolution and signal-to-noise ratio (S/N). All mass spectra reported were obtained in the positive ion mode. The instrument was operated in the linear mode for intact protein detection and the reflectron mode for peptide detection. External calibration was applied to the instrument before data collection. Protein standards (insulin: M+H + = 5734; ubiquitin: M+H+ = 8565; cytochrome C: M+H+ = 12,361 and M+2H2+ = 6181) and peptide calibration standards (bradykinin fragment M+H+ = 757.3997; angiotensin II: M+H+ = 1046.5423; P14R: M+H+ = 1533.8582; ACTH fragment 18-39: M+H+ = 2465.1989; insulin oxidized B: M+H+ = 3494.6513) were used as external calibrations for intact protein and peptide detection, respectively. A typical spectrum was obtained by averaging 3000 laser shots from 30 positions within the sample well. The accelerating voltage was 20 kV. The whole process was controlled by the 4000 Series Explorer Software V3.7.0. Data were processed using Data Explorer Software Version 3.7 (Applied Biosystems, Framingham, MA, USA). The matrixes for MALDI-TOF MS were 15 mg/mL CHCA (for peptides analysis) and 10 mg/mL SA (for proteins analysis) dissolved in 50% ACN containing 0.1% (v/v) TFA. Equivalent amounts (1 µL) of the sample and matrix were 7



sequentially dropped onto the MALDI plate for MALDI-TOF MS analysis.

Preparation of HRP Glycan-Imprinted MNPs. The synthesis procedure of glycan-imprinted MNPs is illustrated in Figure S1. Firstly, Fe3O4@SiO2@DFFPBA MNPs were synthesized and used as the supporting material to further synthesize glycan-imprinted MNPs, as shown in Figure S1A. Then the templates were immobilized on the supporting material through an in situ dual enzymatic digestion procedure. The synthetic route is shown in Figure S1B, which was comprised of five steps: 1) immobilization of intact glycoproteins onto Fe3O4@SiO2@DFFPBA MNPs, 2) in situ digestion of the immobilized glycoproteins by trypsin; 3) in situ digestion of the immobilized glycopeptides by pronase E; 4) oriented imprinting; and 5) template removal. First, 20 mg of Fe3O4@SiO2@DFFPBA MNPs were dispersed in 2 mL of 100 mM ammonium bicarbonate containing 500 mM sodium chloride buffer solution (pH 8.5) by ultrasonication. Then, 1 mg of HRP was added to the suspension and shaken at room temperature for 2 h. The obtained HRP immobilized Fe3O4@SiO2@DFFPBA MNPs were magnetically collected, washed with 100 mM ammonium bicarbonate buffer solution (pH 8.5) and redispersed with 20 mL of the washing buffer. After that, 200 μL of 1 mg / mL trypsin solution were added to the suspension and shaken at 37 °C for 3 h. The obtained long glycopeptides immobilized Fe3O4@SiO2@DFFPBA MNPs were magnetically collected and washed with 100 mM ammonium bicarbonate buffer solution (pH 8.5). The procedure of the second in situ digestion was the same as the first one, except that trypsin was replaced by pronase E. To imprint glycopeptides on the MNPs, the collected glycopeptides bound MNPs were dispersed into 160 mL anhydrous ethanol containing 2.8 mL ammonium hydroxide. 8


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Then 40 mL of TEOS in anhydrous ethanol (10 mM) was added to the suspension and the mixture was gently stirred at room temperature for a period of time. To optimize the thickness of the MIP layer, the time of hydrolysis of TEOS were set at different time. The HRP glycan-imprinted MNPs were collected by a magnet, washed with ethanol for three times, and then dried at 40 °C overnight. To remove the glycopeptides templates, 20 mg glycan-imprinted MNPs were dispersed into 2 mL of 100 mM acetic acid and shaken for 20 min at room temperature. The above washing procedure was repeated three times. After removing the templates, the HRP glycan-imprinted MNPs were magnetically collected, washed with ethanol for three times and then dried at 40 °C overnight.

Non-imprinted MNPs were prepared using the same procedure except for the absence of intact glycoproteins immobilization step. The resulting non-imprinted MNPs were MNPs coated with a silica shell of the same thickness as that of the glycan-imprinted MNPs. Preparation of AHSG glycan-imprinted MNPs. For synthesis of AHSG glycan-imprinted MNPs, to immobilize the template, 20 mg Fe3O4@SiO2@DFFPBA MNPs were dispersed in 2 mL of 100 mM phosphate buffer containing 1 M sodium chloride (pH 7.4). The rest steps were all the same as the HRP glycan-imprinted MNPs. To ensure all glycans can be imprinted with appropriate length coverage, the shortest glycan of AHSG was used to decide the imprinting. According to its molecular length, the imprinting time was set at 35 min, which gives an estimated length coverage of 86.8% for the shortest glycan (Table 1). Corresponding non-imprinted MNPs were synthesized by using the same procedure except for the absence of template immobilization step. Selectivity test of glycan-imprinted MNPs. The selectivity of the HRP glycan-imprinted MNPs 9



was evaluated using HRP, RNase B, TRF, RNase A, and BSA. First, a solution for each protein of 1 mg/mL was separately prepared with 100 mM ammonium bicarbonate buffer containing 500 mM sodium chloride (pH 8.5). Then equivalent HRP glycan-imprinted MNPs and non-imprinted MNPs with different polymerization time (2 mg each) were added to 200 μL of the protein solutions in 250-μL plastic centrifugal tubes, respectively. The tubes were shaken on a rotator at room temperature for 2 h. The MNPs were collected at the tube wall by applying a magnet to the tube wall and rinsed with 200 μL of 100 mM ammonium bicarbonate buffer containing 500 mM sodium chloride (pH 8.5) and 100 mM ammonium bicarbonate buffer (pH 8.5) for three times each. After washing, the MNPs were re-suspended and eluted in 20 μL of 100 mM acetic acid solution for 1 h on a rotator. Finally, the MNPs were trapped to the tube wall again and the eluates were collected by pipetting carefully. Considering TRF is a sialylated glycoprotein, the extraction and washing buffers were changed to 100 mM phosphate buffer containing 1 M sodium chloride (pH 7.4) and 100 mM phosphate buffer (PH 7.4), respectively. The amounts of proteins captured by the MNPs were determined by measuring the amount of proteins in the eluates in terms of UV absorbance at 214 nm. The measurement was repeated for three times. The selectivity of the AHSG glycanimprinted MNPs in the protein level was evaluated as the same as HRP glycan-imprinted MNPs, except changing the binding buffer and washing buffer to 100 mM phosphate buffer containing 1 M sodium chloride (pH 7.4) and 100 mM phosphate buffer (pH 7.4), respectively.

Furthermore, the selectivity of the HRP glycan-imprinted MNPs in the peptide level was further evaluated with tryptic digest of HRP and RNase B. First, a mixture of tryptic digest of HRP 10


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(1 mg/mL) and RNase B (1 mg/mL) were diluted by 100 mM ammonium bicarbonate buffer (pH 8.5) to 5 ng/μL. Then, equivalent (200 μg) HRP glycan-imprinted MNPs and non-imprinted MNPs were added to 100 μL of digest diluted solution in 250-μL plastic microcentrifugal tubes, respectively. The tubes were shaken on a rotator at room temperature for 2 h. The MNPs were collected at the tube wall by applying a magnet to the tube wall and rinsed with 200 μL of 50 mM ammonium bicarbonate buffer (pH 8.5) for three times each. After washing, the MNPs were resuspended and eluted in 10 μL of 20%ACN/5% TFA solution for 1 h on a rotator. Finally, the MNPs were trapped to the tube wall again and the eluates were collected by pipetting carefully. The eluates were analyzed by MALDI-TOF MS. The selectivity of the AHSG glycan-imprinted MNPs in the peptide level was evaluated with tryptic digest of HRP and AHSG. The extraction procedure was all the same as the HRP glycan-imprinted MNPs, except changing the binding buffer and washing buffer to 100 mM phosphate buffer containing 1 M sodium chloride (pH 7.4) and 100 mM phosphate buffer (pH 7.4), respectively. The glycopeptides extracted by the AHSG glycanimprinted MNPs were desialylated after release from the materials. Selective extraction of AHSG from human serum. To evaluate the selective binding ability of the AHSG glycan-imprinted MNPs within complex samples, selective extraction of AHSG from human serum was investigated. First, human serum was diluted 20 times with 100 mM phosphate buffer containing 1 M sodium chloride (pH 7.4). Then equivalent AHSG glycan-imprinted MNPs or non-imprinted MNPs (2 mg) were added to 200 μL of diluted human serum solution in 250-μL plastic centrifugal tubes, respectively. The tubes were shaken on a rotator for 2 h at room 11



temperature. The MNPs were collected at the tube wall by applying a magnet to the tube wall and rinsed with 200 μL of 100 mM phosphate buffer containing 1 M sodium chloride (pH 7.4) and 100 mM phosphate buffer (pH 7.4) for three times each. After washing, the MNPs were re-suspended and eluted in 20 μL of 100 mM acetic acid solution for 1 h on a rotator. Finally, the MNPs were trapped to the tube wall again and the eluates were collected by pipetting carefully. The eluates were directly analyzed by MALDI-TOF MS.

RESULTS AND DISCUSSION

Characterization of MNPs. The selectivity of the boronate affinity MNPs was validated in Figure S2 and S3, which show that sugar-containing species including adenosine and glycoproteins were preferentially captured whereas their non-sugar-containing analogs were largely excluded. Fourier transform infrared (FT-IR) spectrometry, magnetization curve and transmission electron microscopic (TEM) characterization of amino-functionalized, boronic acid-functionalized, imprinted and non-imprinted MNPs are shown in Figure S4 and S5. The saturation magnetization of the imprinted MNPs was about 30 emu/g, which indicates the superparamagnetic property. All the MNPs were well shaped with a diameter of about 100 nm.

Method validation. Magnetic nanoparticles (MNPs) were selected as a substrate because easy magnetic separation favours the ISDED procedure, which involves multiple separation steps. It should be noted that other types of substrates are also applicable. There are two critical issues in 12


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this approach. On one hand, the choice of appropriate enzymes is the first priority. Herein, trypsin was firstly employed to cut the immobilized glycoproteins into long glycopeptides, because trypsin has been widely used in proteomic studies. However, trypsin has only two cleavage sites (lysine and arginine), which usually leads to long peptide chains in the obtained glycopeptides. A long peptide chain on the immobilized template is unfavourable for the imprinting of only the glycan chain since the peptide chain may get involved in the imprinting. To overcome this issue, pronase E was used for a secondary in situ digestion. Pronase E, as a less specific enzyme, preferentially cuts a long peptide into smaller pieces between two neighbouring hydrophobic amino acid residues (Ala, Val, Leu, Ile, Phe, Trp and Tyr). Thus, this dual enzymatic digestion can greatly reduce possible side effects of long peptides in the subsequent imprinting procedure. The working conditions of the above two enzymes are compatible with the boronate affinity binding pH (usually ≥ 8.5), which permits in situ digestion of the glycoprotein captured on the boronate affinity substrate. Besides, both the enzymes are commercially available with low price, which makes this approach cost-efficient. On the other hand, precision imprinting with appropriate nature and thickness for the imprinting layer is another critical aspect. To this end, TEOS polycondensation was employed for the imprinting. The hydrophilic nature of resulting silica layer is favorable for glycan binding. More importantly, a silica layer with appropriate thickness can be precisely generated to imprint just the glycan portion rather than the entire glycopeptide immobilized on the substrate.

Two typical glycoproteins, horseradish peroxidase (HRP) and alpha 2-Heremans-Schmid 13



glycoprotein (AHSG), were selected as the glycan source for preparing alternative templates. HRP possesses 9 N-linked glycosylation sites, each site attached with an identical complex glycan. Due to the presence of an 1→3 fucose on the glycan, PNGase F, which is widely used to digest Nglycans from glycoproteins, fails to cleave the glycan from HRP. AHSG contains 2 N-linked glycosylation sites and 4 O-linked glycosylation sites attached with sialylated glycans (see glycan structures in Figure S6). We first validated the proposed approach for the imprinting of HRP glycan (only one glycan), and then expanded the developed approach for more challenging imprinting of AHSG glycans, which involves simultaneous imprinting of multiple glycans of different molecular lengths and structures.

The in situ dual enzymatic digestion of HRP was validated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). As shown in Figure 2 and Table S1, after the glycoprotein captured by the boronic acid-functionalized MNPs was treated with trypsin, 5 glycopeptides (molecular weight within 2,500–5,000 Da, with a peptide chain containing 19-23 amino acid residues) along with 10 non-glycopeptides were identified in the supernatant. After further treatment with pronase E, 6 non-glycopeptides with low molecular weight (

Precision Imprinting of Glycopeptides for Facile Preparation of Glycan

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