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Facile synthesis of layered mesoporous covalent organic polymers for highly selective enrichment of N-glycopeptides
Accepted Manuscript Facile Synthesis of Layered Mesoporous Covalent Organic Polymers for Highly Selective Enrichment of N-glycopeptides Fengjuan Ding, Zhanying Chu, Quanqing Zhang, Haiyan Liu, Weibing Zhang PII:
S0003-2670(19)30030-3
DOI:
https://doi.org/10.1016/j.aca.2018.12.063
Reference:
ACA 236507
To appear in:
Analytica Chimica Acta
Received Date: 31 October 2018 Revised Date:
22 December 2018
Accepted Date: 24 December 2018
Please cite this article as: F. Ding, Z. Chu, Q. Zhang, H. Liu, W. Zhang, Facile Synthesis of Layered Mesoporous Covalent Organic Polymers for Highly Selective Enrichment of N-glycopeptides, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2018.12.063. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fengjuan Ding,a Zhanying Chu,a Quanqing Zhang,b Haiyan Liu,*, a and Weibing Zhang,*, a
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Facile Synthesis of Layered Mesoporous Covalent Organic Polymers for Highly Selective Enrichment of N-glycopeptides
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Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, People’s Republic of China b Department of Chemistry and the State Key Laboratory of Molecular Engineering, Fudan University, Shanghai 200433, People’s Republic of China
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ABSTRACT: Hydrophilic interaction liquid chromatography is a significant strategy for the separation and enrichment of glycoproteins and glycopeptides. A layered imine-based covalent organic polymer with mesopores (denoted as p-TpBDH) was successfully fabricated by a facile solvothermal method. Then p-TpBDH-OH with abundant hydrophilic groups was evolved from p-TpBDH by the direct reduction. 36 and 40 glycopeptides were identified from IgG digests respectively by p-TpBDH and p-TpBDH-OH. Furthermore, the p-TpBDH-OH exhibits superior selectivity (IgG: BSA=1: 250) for glycopeptides compared with the p-TpBDH. Encouragingly, a total of 463 glycopeptides assigned to 173 glycoproteins were finally identified from only 2 µL human serum by the p-TpBDH-OH. Compared with p-TpBDH, abundant hydrophilic and nitrogen-containing affinity sites of p-TpBDH-OH facilitate effective hydrophilic interaction between the polymeric material and glycopeptides. All the results demonstrate the functionalized hydrophilic covalent organic polymer has great potential in large-scale N-glycoproteomic research.
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Keywords: hydrophilic interaction chromatography; glycopeptide enrichment; solvothermal interaction; mesoporous covalent organic polymer
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Introduction
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Protein N-glycosylation, one of the most prevalent and important post-translational modifications, is of great vital importance in regulating various physiological processes.[1,2] It has been reported that many human-related diseases are associated with the abnormal expression of protein glycosylation and structural changes of sugar chains.[3,4] Therefore, developing a highly selective, sensitive and precise identification strategy for protein glycosylation has a great significance in discovering new clinical biomarkers and clinical diagnosis.[5,6] However, low abundance and serious signal suppression caused by high-abundance nonglycopeptides in biological samples make it difficult to identify glycopeptides directly based on MS analysis. Hence, prior to MS analysis, the high selectivity and efficient enrichment procedure are indispensably required. Currently, a variety of analytical methods are available to capture glycopeptides, including lectin affinity chromatography,[7,8] boric acid affinity chromatography,[9] hydrazide chemistry[10] and hydrophilic interaction liquid chromatography[11]. Wherein hydrophilic interaction liquid chromatography has been widely used as an important sample pretreatment method for glycopeptides enrichment due to its high selectivity, good repeatability and high efficiency.[12–14] In view of that, many functional modification methods have been developed to increase the hydrophilicity of materials such as the surface modification with polysaccharide, L-cysteine, polyethylene glycol.[15– 17] Porous materials have drawn great attention for their specific properties and functions that are impossible to attain in non-porous materials such as high specific area, large pore volume and easily modified surface. In the past decades, several distinct classes of porous materials have rose to be general trends in structural evolution: from inorganic to organic components, from small to large pores, from rigid frameworks to soft dynamic skeletons.[18–21] Among them,
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ACCEPTED MANUSCRIPT mesoporous organic materials are greatly promising in the analysis of real biosamples, because except for modifiable channels, the mesopores also endowed materials size-exclusion effect towards biomacromolecules.[22–28] Covalent organic framework materials (COFs) are crystal porous polymeric materials.[29] Schiff-base condensation reaction which forms a conjugated imine bond from an amine and an aldehyde group has been widely explored in the synthesis of covalent organic frameworks.[30,31] Normally, -OH functionalities adjacent to the Schiff base centers were introduced in COFs to undergo the irreversible proton tautomerism, which made COFs stable in acid, base, and water.[32,33] Thus, we envision that such a reaction will be a promising methodology for the synthesis of polyimine-based porous conjugated polymers with specific hydrophilicity for the application of proteomics. In this work, the hydrophilicity of the layered imine-based p-TpBDH was mainly caused by a few carbonyl groups and hydroxyl groups inside its channels. We believed these affinity groups would facilitate hydrophilic interaction between the polymeric material and glycopeptides. In order to further enhance its capacity of capturing glycopeptides, the p-TpBDH was directly reduced to p-TpBDH-OH. After reduction, the p-TpBDH-OH polymer was evolved with an enhanced hydrophilicity due to the presence of massive hydroxyl and secondary amine groups. Therefore, with the specific mesoporous structure and massive valuable functional groups, the p-TpBDH-OH polymer was used to selectively enrich N-glycopeptides from biosamples.
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Experiment
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Materials and Chemical. 1,3,5-Triformylphloroglucinol (Tp) and pyromellitic-N, N’-bisaminoimide were prepared according to literature procedures[34]. Trypsin, 2,5-dihydroxybenzoic acid (DHB), bovine serum albumin (BSA), immunoglobulin G (IgG), didthiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate and urea were purchased from Sigma-Aldrich (St. Louis, MO, USA). PNGase F was purchased from New England Biolabs (Ipswich, MA). Acetonitrile and trifluoroacetic acid (TFA) were of chromatographic grade and purchased from Aladdin (Shanghai, China). Human serum samples were provided by the Second Affiliated Hospital of Dalian Medical University. Deionized water used in experiments was purified by Milli-Q System (Millipore, Bedford, MA.) All other chemicals and reagents are of analytical grade.
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Synthesis of layered p-TpBDH and p-TpBDH-OH. p-TpBDH was synthesized in a reaction tube (10 mL). 0.3 mmol of 1,3,5-Triformylphloroglucinol (Tp) and 0.45 mmol of pyromellitic-N, N’-bisaminoimide (BDH) were dissolved in the mixture of 3 mL N, N-dimethylacetamide, 1.5 mL dioxane and 0.5 mL 6 mol L-1 aqueous acetic acid. The resulting suspension was sonicated for 5 minutes to get a homogeneous dispersion, and then heated at 120 °C for 3 days. The light red insoluble powder was collected and ultrasonically washed with DMF to obtain the layered p-TpBDH polymer. Then, 1 mmol of p-TpBDH and 4 mmol of sodium borohydride was dissolved in 10 mL of ethanol, and kept at 0 °C for 12 h. A dark red colored p-TpBDH-OH power was finally obtained.
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ACCEPTED MANUSCRIPT Fig. 1. Schematic illustration of synthesis routes of the p-TpBDH and p-TpBDH-OH polymeric materials
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Characterization of layered p-TpBDH and p-TpBDH-OH nanomaterials. The morphology of nanomaterials was observed by transmission electron microscopy (TEM, JEM-2100 EX) and field emission scanning electron microscopy (FE-SEM, Nova Nano SEM 450). Fourier transform infrared (FT-IR) spectra were taken on a Thermo Nicolet 380 (Nicolet, Wisconsin, USA). The PXRD pattern was recorded on a D/max 2550V X-ray diffractometer (Rigaku, Japan) with a Cu Kα source. The scanning range of wide-angle diffraction (2 Theta) was 3–50° and the small angle diffraction was scanned at the range of 0.5–10°. The specific surface area and pore diameter were determined by the Brunauer– Emmett–Teller (BET) method and calculated by the Barrett–Joyner–Halenda (BJH) model, respectively. The elemental analysis was measured by Vario ELⅢ (Elementar, Hanau, Germany). Solid state C13 NMR (SS-NMR) was taken in a Bruker 300 MHz NMR spectrometer.
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Tryptic digestion of the standard proteins and human serum. 1 mg IgG was dissolved in 100 µL of ammonium bicarbonate (50 mmol L-1) containing 8 mol L-1 urea. The mixture was incubated at 60 °C for 40 min after adding 0.15 mg DTT. Then, 0.72 mg IAA was added into the solution. The resulting solution was placed in the dark for 30 min. Finally, Trypsin was added at an enzyme to protein ratio of 1:25 (w/w) and the mixture was incubated at 37 °C for 17 h. For digestion of human serum, 2 µL human serum was dissolved in 18 µL of ammonium bicarbonate (50 mmol L-1) containing 8 mol L-1 urea. The mixture was reduced by 0.03 mg DTT at 60 °C for 45 min and then alkylated by 0.15 mg IAA at room temperature for 30 min. Trypsin was added into the mixture at an enzyme to protein ratio of 1:25 (w/w). The tryptic digestion was carried out at 37 °C for 18 h.
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N-glycopeptides enrichment. IgG tryptic digests were diluted to the desired concentration with the loading buffer. A certain amount of p-TpBDH-OH was added into 200 µL loading buffer and then incubated for 30 min. The supernatant was removed by centrifugation. The p-TpBDH-OH polymer was washed with loading buffer for three times to remove nonspecifically absorbed nonglycopeptides. At last, glycopeptides were released from nanomaterials in 20 µL of the eluting buffer (5% TFA, 30% ACN–H2O). The eluate was directly analyzed by MALDI-TOF MS. The separation and enrichment process is shown in Fig. S1 (supporting information).
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For the enrichment of glycopeptides from human serum, 2 µL human serum lyophilized tryptic digests was redissolved in 400 µL loading buffer (4% TFA, 88% ACN–H2O). 400 µg of p-TpBDH-OH polymers was added into the solution. After incubation for 30 min at the room temperature, the supernatant was discarded by centrifugation. Then the resulting p-TpBDH-OH polymer was rinsed for three times with 400 µL of loading buffer and eluted three times with 100 µL eluting buffer. Lastly, lyophilized eluent was redissolved in the mixture of 17 µL of H2O, 2 µL of glycan buffer and 1 µL PNGase F (500U), and then incubated at 37 °C for 17 h to remove glycans. The resulting peptides were further analyzed by nano LC-MS/MS.
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MALDI-TOF MS analysis. All the MALDI-TOF MS analyses were performed on a 5800 MALDI-TOF/TOF (AB Sciex, USA) with a Nd/YAG laser emitting at 355 nm. 0.5 µL eluent was dropped on the plate and dried at room temperature, then 0.5 µL matrix (DHB, 20 mg mL−1, ACN/H2O/H3PO4 =70:29:1, v/v) was added to the sample spot and dried for MS analysis. The laser pulse frequency and acceleration voltage were set as 200 Hz and 20 kV, respectively.
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Nano LC-MS/MS analysis. Nano LC-MS/MS analysis was performed on an EASY-nLC 1000 nano HPLC system (Thermo Fisher Scientific, Bremen, Germany) connected to a quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The mobile phase was composed of mobile A (0.1% FA/H2O) and mobile phase B (0.1% FA/ACN). The deglycopeptides were resuspended in 10 µL 0.1% FA/H2O, then 1 µL of the solution was loaded on the trap column (Acclaim PepMapC18, 100 µm × 2 cm i.d.). Analytes were separated on the analytical column (Acclaim PepMapC18, 50 µm ×15 cm) with a linear gradient from 2% B to 35% B in 75 min at a flow rate of 300 nL min-1. The column temperature was maintained at 40 °C.
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Q-Exactive plus mass spectrometry was operated in data-dependent MS/MS acquisition mode. The ESI injection voltage was 2.0 kV. The full scan range was from 350 to 1600 m/z with a mass resolution of 70,000. For MS/MS, ions with charge states of 2+, 3+, 4+ were sequentially fragmented by high energy collision dissociation (HCD) with a collision energy (NCE) of 30% and a resolution of 17500. The automatic gain control (AGC) was set to 200000 and the maximum injection time is 50 ms.
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Data searching and analysis. The MS raw data were extracted by Proteome Discoverer software (Thermo Fisher Scientific, version 2.1) and analyzed via Mascot (Matrix Science, London, UK; version 2.3) against Human
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UniProtKB. The parameters for data searching were as follows: two missed cleavages were allowed by trypsin digestion; the mass tolerance of the precursor ions was 10 ppm and the fragment ion mass tolerance was 0.05 Da; peptide level false discovery rate (FDR) was lower than 1%; oxidation on methionine, deamination on asparagine were set as variable modifications and carbamidomethyl on cysteine was set as the fixed modification; N-glycosylation sites were observed at sequences of N-X-S/T (X≠P).
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Results and discussion Synthesis and characterization of p-TpBDH and p-TpBDH-OH nanomaterials. The synthetic routes of p-TpBDH and p-TpBDH-OH are shown in Fig. 1. Briefly, the p-TpBDH was synthesized by schiff-base condensation. Then the successive layered polymer p-TpBDH was physically exfoliated by sonicating in DMF. It was reported that physical exfoliation of synthetic layered crystals has been recently found to be an effective route for the preparation of thick 2D polymers.[35,36] TpBDH-OH with dark red color was finally prepared by reduction (Fig. S2, supporting information). The overall preparation of the p-TpBDH-OH is simple and easily controlled.
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The morphology of as-synthesized p-TpBDH and p-TpBDH-OH was characterized by TEM and FE-SEM (Fig. 2). p-TpBDH and p-TpBDH-OH are porous monodisperse nanomaterials. The sheet structure in gray and white colors show the pore channels of the p-TpBDH and p-TpBDH-OH (Fig. 2a and b). The Aggregated layered structure of p-TpBDH could be apparently observed from FE-SEM (Fig. 2c). There is no distinct change in the SEM images between the p-TpBDH-OH and p-TpBDH (Fig. 2d). The p-TpBDH was linked by imines, which contributes partially with the adjacent benzene ring to form a stable π-stack layered structure.
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Determined FT-IR spectra and C13 sNMR were used to investigate the bond formation and indicate the successful synthesis of p-TpBDH and p-TpBDH-OH covalent organic polymers. A strong peak for -C=C at 1593 cm-1 merged with -C=O stretching frequency 1619 cm-1 appears owing to enol to keto tautomerism present in p-TpBDH. The appearance of two peaks at 1454 and 1283 cm-1 corresponds to the aromatic -C=C and -C-N bond in the keto form of p-TpBDH (Fig. 2e). By contrast, after reduction, the stretching frequency of -OH at 3420 cm-1 increases evidently, and the vibration peaks of -C-O and -C-N bonds (1132 cm-1, 1001 cm-1 and 1283 cm-1) are also significantly enhanced. Further support for the local structure of imine based covalent organic polymer p-TpBDH and the reduced product p-TpBDH-OH are given by C13 solid state NMR spectroscopy (Fig. 2f). p-TpBDH shows a clear signal at 178.4 ppm corresponding to the carbonyl (-C=O) carbons of the keto form. However, the resonance of carbonyl carbons of the BDH linker appears at 159.0 ppm. The peak of 146.2 ppm confirms the presence of the -C-N bond, instead of the -C=N bond, which would have been a signature peak if TpBDH had been existing in the enol form (~160 ppm). After reduction, the disappearance of 159.0 ppm and the high intensity of 40.6 ppm indicates the successful reduction from imine to the second amine. The appearance of 22.4 ppm and 16.9 ppm with high intensity demonstrates the reduction of carbonyl groups.
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The PXRD patterns of p-TpBDH and p-TpBDH-OH shows a broad peak at 2θ ~30° due to the 001 plane reflection (Fig. S3, supporting information). The broad peak indicates the π stacking between successive layers of the conjugated covalent organic polymers. The peak at minor 2θ ~5° corresponds to the reflection from 100 plane and the 2θ ~8.7° indicates the layered p-TpBDH and p-TpBDH-OH polymers have been successfully prepared. The small peak at 2θ ~0.7° determined from the small-angle diffraction indicates the covalent organic polymer p-TpBDH is a periodically ordered mesoporous polymeric material (Fig. S4, supproting information). Elemental analysis was used to accurately determine the elemental composition of the two polymers. No distinct content change of C, H, O was determined as a result of the simple reductive hydrogenation of the conversation from p-TpBDH to p-TBDH-OH (Table S1).
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The porosity characteristics of p-TpBDH and p-TpBDH-OH were determined by nitrogen adsorption–desorption isotherms (Fig. S5a and c, supporting information). The BET surface area and pore volume of p-TpBDH are calculated to be 93.25 m2 g-1 and 0.39 cm3 g-1, while 64.04 m2 g-1 and 0.25 cm3 g-1 for p-TpBDH-OH (Fig. S5b and d, supporting information), respectively. The minor pore size of p-TpBDH-OH is identical to p-TpBDH with similar 3.72 nm calculated by BJH model. The broad peak with a wide range of 20–100 nm clearly determined in the p-TpBDH and p-TpBDH-OH is originated from interparticle space. Large interlayer space facilitates glycopeptides mass transportation, allowing them to interact adequately with hydrophilic groups inside the mesopores. [37, 38]
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All the data above prove that the imine-based polymer p-TpBDH and the reductive polymer p-TpBDH-OH were successfully fabricated. Specially, the enhanced hydrophilic property of the p-TpBDH-OH compared with p-TpBDH would significantly promote the capability for the enrichment of glycopeptides with high efficiency and selectivity.
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Fig. 2. TEM images of (a) p-TpBDH and (b) p-TpBDH-OH; FE-SEM images of (c) p-TpBDH and (d) p-TpBDH-OH; (e) IR spectra of p-TpBDH and p-TpBDH-OH; (f) 13C sNMR spectra of p-TpBDH and p-TpBDH-OH.
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Optimization and Selective enrichment of N-glycopeptides from the tryptic digest of a standard protein.
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Without enrichment, merely five glycopeptides were determined with weak intensity because of the severe suppression of abundant nonglycopeptides present in tryptic digests (Fig. S11, supporting information). However, 36 and 40 glycopeptides were detected with obviously improved S/N ratios and a clean background after enrichment by p-TpBDH and p-TpBDH-OH, respectively (Fig. 3a and b). The detailed information of the identified glycopeptides is shown in Table S2. Apparently, the mesoporous p-TpBDH-OH polymer exhibits excellent enrichment performance for tryptic digests of standard glycoprotein (IgG).
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The enrichment condition of glycopeptides based on HILIC method is significantly important. To get an optimal enrichment, we first examined the enrichment performance of the two polymeric materials in different loading buffers. The acidity of loading buffer was increased from 0.1% TFA to 6% TFA. For p-TpBDH-OH, only several glycopeptides were observed with low intensity from 0.1% to 2% TFA. However, the number of glycopeptides increased clearly with the increasing acidity from 3% to 6% TFA (Fig. S6, supporting information). When the concentration of TFA was 5%, the optimal enrichment efficiency for glycopeptides was obtained. Similarly, for p-TpBDH, the optimal acidity of loading buffer was also 5% TFA (Fig.S7, supporting information). Then glycopeptides of tryptic digest of IgG were captured in a wide range concentration of acetonitrile from 80% ACN to 94% ACN. Considering the number and intensity of glycopeptides detected in mass spectra, the optimal loading conditions for p-TpBDH-OH was ACN/H2O/TFA=94/1/5 (Fig. S8, supporting information), but for p-TpBDH the concentration of acetonitrile was as low as 85% (Fig. S9, supporting information). Based on hydrophilic chromatography, the optimal acetonitrile concentration of p-TpBDH-OH was higher than the p-TpBDH because of the enhanced hydrophilicity of the p-TpBDH-OH material. Furthermore, adsorption capability of p-TpBDH-OH and p-TpBDH was investigated. Different amounts of materials were used to capture glycopeptides from 2 µL IgG digests (1mg mL-1). Three glycopeptides with m/z 2634, 2797, and 2959 were selected as detected markers. The signal intensities of the three glycopeptides all reached the maximum values when the amount of materials was up to 0.4 mg (Fig. S10, supporting information). However, the signal intensity of p-TpBDH-OH is obviously higher than the p-TpBDH indicating that p-TpBDH-OH should have a better enrichment performance for glycopeptides than p-TpBDH.
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Fig. 3. MALDI-TOF mass spectra of the tryptic digest of IgG (1.67 ×10-7 mol L-1, 200 µ L). After enrichment by (a) p-TpBDH and (b) p-TpBDH-OH.
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Generally, the capacity of capturing glycopeptides from thimbleful samples is another important criterion for evaluating the enrichment performance of a material. When the concentration of IgG tryptic digest was 7.5 fmol µL-1, 10 and 14 glycopeptides could be easily detected by p-TpBDH and p-TpBDH-OH respectively (Fig. S13, parts a and b). Coincidently, 3 and 6 glycopeptides could still be identified in 1.5 fmol µL-1 digests (Fig. S13, parts c and d) after enrichment by the p-TpBDH and p-TpBDH-OH, respectively. Therefore, the p-TpBDH and p-TpBDH-OH demonstrate equally excellent sensitivity to glycopeptides. The detection limits of the two kinds of nanomaterials are as low as 1.5 fmol µL-1. However, compared with p-TpBDH, more glycopeptides with higher intensity were observed in the mass spectra when the same minor amount digests were enriched by the p-TpBDH-OH. Therefore, the p-TpBDH-OH nanomaterial shows high sensitivity towards glycopeptides.
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Fig. 4. MALDI-TOF mass spectra of tryptic digest mixture of IgG (1×10-7 mo L-1) and BSA (IgG:BSA=1:250, mol/mol) after enrichment by the TpBDH-OH nanomaterial.
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Inspired by the outstanding performance of p-TpBDH-OH, nano LC-MS/MS was used to investigate its enrichment performance for low-abundance glycopeptides in real biosamples. 2 µ L of human serum was enriched by p-TpBDH-OH following the same protocol for glycopeptides enriched from standard proteins. A total of 463 glycopeptides belonging to 173 glycoproteins were identified from 2 µ L of human serum within three independent replicates (Fig. 5). Detailed information regarding the identified N-glycopeptide is shown in Table S3. However, merely 243 glycopeptides belonging to 86 proteins were identified from the same amount human serum by p-TpBDH within three independent replicates. Recently, many functionalized porous materials were synthesized and successfully applied for glycopeptide enrichment (See in Table S4). Comparatively, our determined results demonstrate the
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Fig. 5. Identified (a) N-glycopeptides; (b) glycoproteins from human serum using p-TpBDH-OH.
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Conclusion
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Conflicts of interest
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Acknowledgments
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Supporting Information
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References
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In summary, a mesoporous covalent organic polymer p-TpBDH-OH was successfully synthesized through the direct reduction of imine-based mesoporous polymer p-TpBDH. Owing to the massive hydrophilic and nitrogen-containing affinity sites inside channels, p-TpBDH-OH exhibits excellent enrichment capacity with remarkably high selectivity and sensitivity towards glycopeptides in biological samples. This work greatly complements the application of imine-based covalent organic polymers for glycoproteomics, and the excellent enrichment performance demonstrated the prominent potential application of this kind of material for proteomics and disease diagnosis. The authors declare no conflicts of interest.
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This research was financially supported by the National Natural Science Foundation of China (No.21475044, 51472121), the National Key Technology R&D Program (2015BAK44B00), the National Key Scientific Instrument and Equipment Development Project (No. 2012YQ004403), and the Fundamental Research Funds for the Central Universities (222201817022).
This material includes figures of characteristics of polymeric materials; figures of MALDI-TOF mass spectra of glycopeptides enriched from IgG tryptic digests by p-TpBDH and p-TpBDH-OH; table of N-glycopeptides of IgG tryptic digests identified by MALDI-TOF; tables of identified N-glycopeptides determined by nano LC-MS/MS from 2µ L of human serum tryptic digests enriched by p-TpBDH-OH and p-TpBDH (PDF).
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Sun, Hydrophilic probe in mesoporous pore for selective enrichment of endogenous glycopeptides in biological samples, Anal. Chim. Acta 1024 (2018) 84–92. (25) J. Li, J. Wang, Y. Ling, Z. Chen, M. Gao, X. Zhang, Y. Zhou, Unprecedented highly efficient capture of glycopeptides by Fe3O4@Mg-MOF-74 core-shell nanoparticles, Chem. Commun. 53 (2017) 4018–4021. (26) J. Wang, J. Li, M. Gao, X. Zhang, Self-assembling covalent organic framework functionalized magnetic graphene hydrophilic biocomposites as an ultrasensitive matrix for N-linked glycopeptide, Nanoscale 9 (2017) 10750–10756. (27) Z. Wang, R. Wu, H. Chen, N. Sun, C. Deng, Synthesis of zwitterionic hydrophilic magnetic mesoporous silica materials for endogenous glycopeptide analysis in human saliva, Nanoscale 10 (2018) 5335–5341. (28) Y. Ma, F. Yuan, X. Zhang, Y. Zhou, X. Zhang, Highly efficient enrichment of N-linked glycopeptides using a hydrophilic covalent-organic framework, Analyst 142 (2017) 3212–3218. (29) A.P. Côté, A.I. 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Hightlights The layered covalent organic polymer p-TpBDH-OH with mesopores was fabricated by a facile reduction of p-TpBDH. TpBDH-OH exhibit excellent performance and high selectivity for capturing glycopeptides from complicated biosamples due to its enhanced hydrophilicity and specific porous structure.
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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0003-2670(19)30030-3
DOI:
https://doi.org/10.1016/j.aca.2018.12.063
Reference:
ACA 236507
To appear in:
Analytica Chimica Acta
Received Date: 31 October 2018 Revised Date:
22 December 2018
Accepted Date: 24 December 2018
Please cite this article as: F. Ding, Z. Chu, Q. Zhang, H. Liu, W. Zhang, Facile Synthesis of Layered Mesoporous Covalent Organic Polymers for Highly Selective Enrichment of N-glycopeptides, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2018.12.063. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fengjuan Ding,a Zhanying Chu,a Quanqing Zhang,b Haiyan Liu,*, a and Weibing Zhang,*, a
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Facile Synthesis of Layered Mesoporous Covalent Organic Polymers for Highly Selective Enrichment of N-glycopeptides
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Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, People’s Republic of China b Department of Chemistry and the State Key Laboratory of Molecular Engineering, Fudan University, Shanghai 200433, People’s Republic of China
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ABSTRACT: Hydrophilic interaction liquid chromatography is a significant strategy for the separation and enrichment of glycoproteins and glycopeptides. A layered imine-based covalent organic polymer with mesopores (denoted as p-TpBDH) was successfully fabricated by a facile solvothermal method. Then p-TpBDH-OH with abundant hydrophilic groups was evolved from p-TpBDH by the direct reduction. 36 and 40 glycopeptides were identified from IgG digests respectively by p-TpBDH and p-TpBDH-OH. Furthermore, the p-TpBDH-OH exhibits superior selectivity (IgG: BSA=1: 250) for glycopeptides compared with the p-TpBDH. Encouragingly, a total of 463 glycopeptides assigned to 173 glycoproteins were finally identified from only 2 µL human serum by the p-TpBDH-OH. Compared with p-TpBDH, abundant hydrophilic and nitrogen-containing affinity sites of p-TpBDH-OH facilitate effective hydrophilic interaction between the polymeric material and glycopeptides. All the results demonstrate the functionalized hydrophilic covalent organic polymer has great potential in large-scale N-glycoproteomic research.
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Keywords: hydrophilic interaction chromatography; glycopeptide enrichment; solvothermal interaction; mesoporous covalent organic polymer
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Introduction
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Protein N-glycosylation, one of the most prevalent and important post-translational modifications, is of great vital importance in regulating various physiological processes.[1,2] It has been reported that many human-related diseases are associated with the abnormal expression of protein glycosylation and structural changes of sugar chains.[3,4] Therefore, developing a highly selective, sensitive and precise identification strategy for protein glycosylation has a great significance in discovering new clinical biomarkers and clinical diagnosis.[5,6] However, low abundance and serious signal suppression caused by high-abundance nonglycopeptides in biological samples make it difficult to identify glycopeptides directly based on MS analysis. Hence, prior to MS analysis, the high selectivity and efficient enrichment procedure are indispensably required. Currently, a variety of analytical methods are available to capture glycopeptides, including lectin affinity chromatography,[7,8] boric acid affinity chromatography,[9] hydrazide chemistry[10] and hydrophilic interaction liquid chromatography[11]. Wherein hydrophilic interaction liquid chromatography has been widely used as an important sample pretreatment method for glycopeptides enrichment due to its high selectivity, good repeatability and high efficiency.[12–14] In view of that, many functional modification methods have been developed to increase the hydrophilicity of materials such as the surface modification with polysaccharide, L-cysteine, polyethylene glycol.[15– 17] Porous materials have drawn great attention for their specific properties and functions that are impossible to attain in non-porous materials such as high specific area, large pore volume and easily modified surface. In the past decades, several distinct classes of porous materials have rose to be general trends in structural evolution: from inorganic to organic components, from small to large pores, from rigid frameworks to soft dynamic skeletons.[18–21] Among them,
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ACCEPTED MANUSCRIPT mesoporous organic materials are greatly promising in the analysis of real biosamples, because except for modifiable channels, the mesopores also endowed materials size-exclusion effect towards biomacromolecules.[22–28] Covalent organic framework materials (COFs) are crystal porous polymeric materials.[29] Schiff-base condensation reaction which forms a conjugated imine bond from an amine and an aldehyde group has been widely explored in the synthesis of covalent organic frameworks.[30,31] Normally, -OH functionalities adjacent to the Schiff base centers were introduced in COFs to undergo the irreversible proton tautomerism, which made COFs stable in acid, base, and water.[32,33] Thus, we envision that such a reaction will be a promising methodology for the synthesis of polyimine-based porous conjugated polymers with specific hydrophilicity for the application of proteomics. In this work, the hydrophilicity of the layered imine-based p-TpBDH was mainly caused by a few carbonyl groups and hydroxyl groups inside its channels. We believed these affinity groups would facilitate hydrophilic interaction between the polymeric material and glycopeptides. In order to further enhance its capacity of capturing glycopeptides, the p-TpBDH was directly reduced to p-TpBDH-OH. After reduction, the p-TpBDH-OH polymer was evolved with an enhanced hydrophilicity due to the presence of massive hydroxyl and secondary amine groups. Therefore, with the specific mesoporous structure and massive valuable functional groups, the p-TpBDH-OH polymer was used to selectively enrich N-glycopeptides from biosamples.
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Experiment
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Materials and Chemical. 1,3,5-Triformylphloroglucinol (Tp) and pyromellitic-N, N’-bisaminoimide were prepared according to literature procedures[34]. Trypsin, 2,5-dihydroxybenzoic acid (DHB), bovine serum albumin (BSA), immunoglobulin G (IgG), didthiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate and urea were purchased from Sigma-Aldrich (St. Louis, MO, USA). PNGase F was purchased from New England Biolabs (Ipswich, MA). Acetonitrile and trifluoroacetic acid (TFA) were of chromatographic grade and purchased from Aladdin (Shanghai, China). Human serum samples were provided by the Second Affiliated Hospital of Dalian Medical University. Deionized water used in experiments was purified by Milli-Q System (Millipore, Bedford, MA.) All other chemicals and reagents are of analytical grade.
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Synthesis of layered p-TpBDH and p-TpBDH-OH. p-TpBDH was synthesized in a reaction tube (10 mL). 0.3 mmol of 1,3,5-Triformylphloroglucinol (Tp) and 0.45 mmol of pyromellitic-N, N’-bisaminoimide (BDH) were dissolved in the mixture of 3 mL N, N-dimethylacetamide, 1.5 mL dioxane and 0.5 mL 6 mol L-1 aqueous acetic acid. The resulting suspension was sonicated for 5 minutes to get a homogeneous dispersion, and then heated at 120 °C for 3 days. The light red insoluble powder was collected and ultrasonically washed with DMF to obtain the layered p-TpBDH polymer. Then, 1 mmol of p-TpBDH and 4 mmol of sodium borohydride was dissolved in 10 mL of ethanol, and kept at 0 °C for 12 h. A dark red colored p-TpBDH-OH power was finally obtained.
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ACCEPTED MANUSCRIPT Fig. 1. Schematic illustration of synthesis routes of the p-TpBDH and p-TpBDH-OH polymeric materials
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Characterization of layered p-TpBDH and p-TpBDH-OH nanomaterials. The morphology of nanomaterials was observed by transmission electron microscopy (TEM, JEM-2100 EX) and field emission scanning electron microscopy (FE-SEM, Nova Nano SEM 450). Fourier transform infrared (FT-IR) spectra were taken on a Thermo Nicolet 380 (Nicolet, Wisconsin, USA). The PXRD pattern was recorded on a D/max 2550V X-ray diffractometer (Rigaku, Japan) with a Cu Kα source. The scanning range of wide-angle diffraction (2 Theta) was 3–50° and the small angle diffraction was scanned at the range of 0.5–10°. The specific surface area and pore diameter were determined by the Brunauer– Emmett–Teller (BET) method and calculated by the Barrett–Joyner–Halenda (BJH) model, respectively. The elemental analysis was measured by Vario ELⅢ (Elementar, Hanau, Germany). Solid state C13 NMR (SS-NMR) was taken in a Bruker 300 MHz NMR spectrometer.
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Tryptic digestion of the standard proteins and human serum. 1 mg IgG was dissolved in 100 µL of ammonium bicarbonate (50 mmol L-1) containing 8 mol L-1 urea. The mixture was incubated at 60 °C for 40 min after adding 0.15 mg DTT. Then, 0.72 mg IAA was added into the solution. The resulting solution was placed in the dark for 30 min. Finally, Trypsin was added at an enzyme to protein ratio of 1:25 (w/w) and the mixture was incubated at 37 °C for 17 h. For digestion of human serum, 2 µL human serum was dissolved in 18 µL of ammonium bicarbonate (50 mmol L-1) containing 8 mol L-1 urea. The mixture was reduced by 0.03 mg DTT at 60 °C for 45 min and then alkylated by 0.15 mg IAA at room temperature for 30 min. Trypsin was added into the mixture at an enzyme to protein ratio of 1:25 (w/w). The tryptic digestion was carried out at 37 °C for 18 h.
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N-glycopeptides enrichment. IgG tryptic digests were diluted to the desired concentration with the loading buffer. A certain amount of p-TpBDH-OH was added into 200 µL loading buffer and then incubated for 30 min. The supernatant was removed by centrifugation. The p-TpBDH-OH polymer was washed with loading buffer for three times to remove nonspecifically absorbed nonglycopeptides. At last, glycopeptides were released from nanomaterials in 20 µL of the eluting buffer (5% TFA, 30% ACN–H2O). The eluate was directly analyzed by MALDI-TOF MS. The separation and enrichment process is shown in Fig. S1 (supporting information).
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For the enrichment of glycopeptides from human serum, 2 µL human serum lyophilized tryptic digests was redissolved in 400 µL loading buffer (4% TFA, 88% ACN–H2O). 400 µg of p-TpBDH-OH polymers was added into the solution. After incubation for 30 min at the room temperature, the supernatant was discarded by centrifugation. Then the resulting p-TpBDH-OH polymer was rinsed for three times with 400 µL of loading buffer and eluted three times with 100 µL eluting buffer. Lastly, lyophilized eluent was redissolved in the mixture of 17 µL of H2O, 2 µL of glycan buffer and 1 µL PNGase F (500U), and then incubated at 37 °C for 17 h to remove glycans. The resulting peptides were further analyzed by nano LC-MS/MS.
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MALDI-TOF MS analysis. All the MALDI-TOF MS analyses were performed on a 5800 MALDI-TOF/TOF (AB Sciex, USA) with a Nd/YAG laser emitting at 355 nm. 0.5 µL eluent was dropped on the plate and dried at room temperature, then 0.5 µL matrix (DHB, 20 mg mL−1, ACN/H2O/H3PO4 =70:29:1, v/v) was added to the sample spot and dried for MS analysis. The laser pulse frequency and acceleration voltage were set as 200 Hz and 20 kV, respectively.
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Nano LC-MS/MS analysis. Nano LC-MS/MS analysis was performed on an EASY-nLC 1000 nano HPLC system (Thermo Fisher Scientific, Bremen, Germany) connected to a quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The mobile phase was composed of mobile A (0.1% FA/H2O) and mobile phase B (0.1% FA/ACN). The deglycopeptides were resuspended in 10 µL 0.1% FA/H2O, then 1 µL of the solution was loaded on the trap column (Acclaim PepMapC18, 100 µm × 2 cm i.d.). Analytes were separated on the analytical column (Acclaim PepMapC18, 50 µm ×15 cm) with a linear gradient from 2% B to 35% B in 75 min at a flow rate of 300 nL min-1. The column temperature was maintained at 40 °C.
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Q-Exactive plus mass spectrometry was operated in data-dependent MS/MS acquisition mode. The ESI injection voltage was 2.0 kV. The full scan range was from 350 to 1600 m/z with a mass resolution of 70,000. For MS/MS, ions with charge states of 2+, 3+, 4+ were sequentially fragmented by high energy collision dissociation (HCD) with a collision energy (NCE) of 30% and a resolution of 17500. The automatic gain control (AGC) was set to 200000 and the maximum injection time is 50 ms.
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Data searching and analysis. The MS raw data were extracted by Proteome Discoverer software (Thermo Fisher Scientific, version 2.1) and analyzed via Mascot (Matrix Science, London, UK; version 2.3) against Human
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UniProtKB. The parameters for data searching were as follows: two missed cleavages were allowed by trypsin digestion; the mass tolerance of the precursor ions was 10 ppm and the fragment ion mass tolerance was 0.05 Da; peptide level false discovery rate (FDR) was lower than 1%; oxidation on methionine, deamination on asparagine were set as variable modifications and carbamidomethyl on cysteine was set as the fixed modification; N-glycosylation sites were observed at sequences of N-X-S/T (X≠P).
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Results and discussion Synthesis and characterization of p-TpBDH and p-TpBDH-OH nanomaterials. The synthetic routes of p-TpBDH and p-TpBDH-OH are shown in Fig. 1. Briefly, the p-TpBDH was synthesized by schiff-base condensation. Then the successive layered polymer p-TpBDH was physically exfoliated by sonicating in DMF. It was reported that physical exfoliation of synthetic layered crystals has been recently found to be an effective route for the preparation of thick 2D polymers.[35,36] TpBDH-OH with dark red color was finally prepared by reduction (Fig. S2, supporting information). The overall preparation of the p-TpBDH-OH is simple and easily controlled.
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The morphology of as-synthesized p-TpBDH and p-TpBDH-OH was characterized by TEM and FE-SEM (Fig. 2). p-TpBDH and p-TpBDH-OH are porous monodisperse nanomaterials. The sheet structure in gray and white colors show the pore channels of the p-TpBDH and p-TpBDH-OH (Fig. 2a and b). The Aggregated layered structure of p-TpBDH could be apparently observed from FE-SEM (Fig. 2c). There is no distinct change in the SEM images between the p-TpBDH-OH and p-TpBDH (Fig. 2d). The p-TpBDH was linked by imines, which contributes partially with the adjacent benzene ring to form a stable π-stack layered structure.
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Determined FT-IR spectra and C13 sNMR were used to investigate the bond formation and indicate the successful synthesis of p-TpBDH and p-TpBDH-OH covalent organic polymers. A strong peak for -C=C at 1593 cm-1 merged with -C=O stretching frequency 1619 cm-1 appears owing to enol to keto tautomerism present in p-TpBDH. The appearance of two peaks at 1454 and 1283 cm-1 corresponds to the aromatic -C=C and -C-N bond in the keto form of p-TpBDH (Fig. 2e). By contrast, after reduction, the stretching frequency of -OH at 3420 cm-1 increases evidently, and the vibration peaks of -C-O and -C-N bonds (1132 cm-1, 1001 cm-1 and 1283 cm-1) are also significantly enhanced. Further support for the local structure of imine based covalent organic polymer p-TpBDH and the reduced product p-TpBDH-OH are given by C13 solid state NMR spectroscopy (Fig. 2f). p-TpBDH shows a clear signal at 178.4 ppm corresponding to the carbonyl (-C=O) carbons of the keto form. However, the resonance of carbonyl carbons of the BDH linker appears at 159.0 ppm. The peak of 146.2 ppm confirms the presence of the -C-N bond, instead of the -C=N bond, which would have been a signature peak if TpBDH had been existing in the enol form (~160 ppm). After reduction, the disappearance of 159.0 ppm and the high intensity of 40.6 ppm indicates the successful reduction from imine to the second amine. The appearance of 22.4 ppm and 16.9 ppm with high intensity demonstrates the reduction of carbonyl groups.
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The PXRD patterns of p-TpBDH and p-TpBDH-OH shows a broad peak at 2θ ~30° due to the 001 plane reflection (Fig. S3, supporting information). The broad peak indicates the π stacking between successive layers of the conjugated covalent organic polymers. The peak at minor 2θ ~5° corresponds to the reflection from 100 plane and the 2θ ~8.7° indicates the layered p-TpBDH and p-TpBDH-OH polymers have been successfully prepared. The small peak at 2θ ~0.7° determined from the small-angle diffraction indicates the covalent organic polymer p-TpBDH is a periodically ordered mesoporous polymeric material (Fig. S4, supproting information). Elemental analysis was used to accurately determine the elemental composition of the two polymers. No distinct content change of C, H, O was determined as a result of the simple reductive hydrogenation of the conversation from p-TpBDH to p-TBDH-OH (Table S1).
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The porosity characteristics of p-TpBDH and p-TpBDH-OH were determined by nitrogen adsorption–desorption isotherms (Fig. S5a and c, supporting information). The BET surface area and pore volume of p-TpBDH are calculated to be 93.25 m2 g-1 and 0.39 cm3 g-1, while 64.04 m2 g-1 and 0.25 cm3 g-1 for p-TpBDH-OH (Fig. S5b and d, supporting information), respectively. The minor pore size of p-TpBDH-OH is identical to p-TpBDH with similar 3.72 nm calculated by BJH model. The broad peak with a wide range of 20–100 nm clearly determined in the p-TpBDH and p-TpBDH-OH is originated from interparticle space. Large interlayer space facilitates glycopeptides mass transportation, allowing them to interact adequately with hydrophilic groups inside the mesopores. [37, 38]
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All the data above prove that the imine-based polymer p-TpBDH and the reductive polymer p-TpBDH-OH were successfully fabricated. Specially, the enhanced hydrophilic property of the p-TpBDH-OH compared with p-TpBDH would significantly promote the capability for the enrichment of glycopeptides with high efficiency and selectivity.
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Fig. 2. TEM images of (a) p-TpBDH and (b) p-TpBDH-OH; FE-SEM images of (c) p-TpBDH and (d) p-TpBDH-OH; (e) IR spectra of p-TpBDH and p-TpBDH-OH; (f) 13C sNMR spectra of p-TpBDH and p-TpBDH-OH.
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Optimization and Selective enrichment of N-glycopeptides from the tryptic digest of a standard protein.
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Without enrichment, merely five glycopeptides were determined with weak intensity because of the severe suppression of abundant nonglycopeptides present in tryptic digests (Fig. S11, supporting information). However, 36 and 40 glycopeptides were detected with obviously improved S/N ratios and a clean background after enrichment by p-TpBDH and p-TpBDH-OH, respectively (Fig. 3a and b). The detailed information of the identified glycopeptides is shown in Table S2. Apparently, the mesoporous p-TpBDH-OH polymer exhibits excellent enrichment performance for tryptic digests of standard glycoprotein (IgG).
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The enrichment condition of glycopeptides based on HILIC method is significantly important. To get an optimal enrichment, we first examined the enrichment performance of the two polymeric materials in different loading buffers. The acidity of loading buffer was increased from 0.1% TFA to 6% TFA. For p-TpBDH-OH, only several glycopeptides were observed with low intensity from 0.1% to 2% TFA. However, the number of glycopeptides increased clearly with the increasing acidity from 3% to 6% TFA (Fig. S6, supporting information). When the concentration of TFA was 5%, the optimal enrichment efficiency for glycopeptides was obtained. Similarly, for p-TpBDH, the optimal acidity of loading buffer was also 5% TFA (Fig.S7, supporting information). Then glycopeptides of tryptic digest of IgG were captured in a wide range concentration of acetonitrile from 80% ACN to 94% ACN. Considering the number and intensity of glycopeptides detected in mass spectra, the optimal loading conditions for p-TpBDH-OH was ACN/H2O/TFA=94/1/5 (Fig. S8, supporting information), but for p-TpBDH the concentration of acetonitrile was as low as 85% (Fig. S9, supporting information). Based on hydrophilic chromatography, the optimal acetonitrile concentration of p-TpBDH-OH was higher than the p-TpBDH because of the enhanced hydrophilicity of the p-TpBDH-OH material. Furthermore, adsorption capability of p-TpBDH-OH and p-TpBDH was investigated. Different amounts of materials were used to capture glycopeptides from 2 µL IgG digests (1mg mL-1). Three glycopeptides with m/z 2634, 2797, and 2959 were selected as detected markers. The signal intensities of the three glycopeptides all reached the maximum values when the amount of materials was up to 0.4 mg (Fig. S10, supporting information). However, the signal intensity of p-TpBDH-OH is obviously higher than the p-TpBDH indicating that p-TpBDH-OH should have a better enrichment performance for glycopeptides than p-TpBDH.
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Fig. 3. MALDI-TOF mass spectra of the tryptic digest of IgG (1.67 ×10-7 mol L-1, 200 µ L). After enrichment by (a) p-TpBDH and (b) p-TpBDH-OH.
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Generally, the capacity of capturing glycopeptides from thimbleful samples is another important criterion for evaluating the enrichment performance of a material. When the concentration of IgG tryptic digest was 7.5 fmol µL-1, 10 and 14 glycopeptides could be easily detected by p-TpBDH and p-TpBDH-OH respectively (Fig. S13, parts a and b). Coincidently, 3 and 6 glycopeptides could still be identified in 1.5 fmol µL-1 digests (Fig. S13, parts c and d) after enrichment by the p-TpBDH and p-TpBDH-OH, respectively. Therefore, the p-TpBDH and p-TpBDH-OH demonstrate equally excellent sensitivity to glycopeptides. The detection limits of the two kinds of nanomaterials are as low as 1.5 fmol µL-1. However, compared with p-TpBDH, more glycopeptides with higher intensity were observed in the mass spectra when the same minor amount digests were enriched by the p-TpBDH-OH. Therefore, the p-TpBDH-OH nanomaterial shows high sensitivity towards glycopeptides.
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Fig. 4. MALDI-TOF mass spectra of tryptic digest mixture of IgG (1×10-7 mo L-1) and BSA (IgG:BSA=1:250, mol/mol) after enrichment by the TpBDH-OH nanomaterial.
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Inspired by the outstanding performance of p-TpBDH-OH, nano LC-MS/MS was used to investigate its enrichment performance for low-abundance glycopeptides in real biosamples. 2 µ L of human serum was enriched by p-TpBDH-OH following the same protocol for glycopeptides enriched from standard proteins. A total of 463 glycopeptides belonging to 173 glycoproteins were identified from 2 µ L of human serum within three independent replicates (Fig. 5). Detailed information regarding the identified N-glycopeptide is shown in Table S3. However, merely 243 glycopeptides belonging to 86 proteins were identified from the same amount human serum by p-TpBDH within three independent replicates. Recently, many functionalized porous materials were synthesized and successfully applied for glycopeptide enrichment (See in Table S4). Comparatively, our determined results demonstrate the
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Fig. 5. Identified (a) N-glycopeptides; (b) glycoproteins from human serum using p-TpBDH-OH.
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References
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In summary, a mesoporous covalent organic polymer p-TpBDH-OH was successfully synthesized through the direct reduction of imine-based mesoporous polymer p-TpBDH. Owing to the massive hydrophilic and nitrogen-containing affinity sites inside channels, p-TpBDH-OH exhibits excellent enrichment capacity with remarkably high selectivity and sensitivity towards glycopeptides in biological samples. This work greatly complements the application of imine-based covalent organic polymers for glycoproteomics, and the excellent enrichment performance demonstrated the prominent potential application of this kind of material for proteomics and disease diagnosis. The authors declare no conflicts of interest.
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This research was financially supported by the National Natural Science Foundation of China (No.21475044, 51472121), the National Key Technology R&D Program (2015BAK44B00), the National Key Scientific Instrument and Equipment Development Project (No. 2012YQ004403), and the Fundamental Research Funds for the Central Universities (222201817022).
This material includes figures of characteristics of polymeric materials; figures of MALDI-TOF mass spectra of glycopeptides enriched from IgG tryptic digests by p-TpBDH and p-TpBDH-OH; table of N-glycopeptides of IgG tryptic digests identified by MALDI-TOF; tables of identified N-glycopeptides determined by nano LC-MS/MS from 2µ L of human serum tryptic digests enriched by p-TpBDH-OH and p-TpBDH (PDF).
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Hightlights The layered covalent organic polymer p-TpBDH-OH with mesopores was fabricated by a facile reduction of p-TpBDH. TpBDH-OH exhibit excellent performance and high selectivity for capturing glycopeptides from complicated biosamples due to its enhanced hydrophilicity and specific porous structure.
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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: