Enrichment Of N-linked Glycopeptides Research Articles

Boronic acid and fructose-1,6-diphosphate dual-functionalized highly hydrophilic Zr-MOF for HILIC enrichment of N-linked glycopeptides.

The HILIC enrichment is a greatly compatible strategy for the extraction of glycopeptides in proteomics. Herein, a boric acid and fructose-1,6-diphosphate (FDP) dual-functionalized Zr-based metal-organic framework material UIO-PBA&FDP (UIO is the abbreviation for the University of Oslo, and PBA is the abbreviation for carboxy phenylboronic acid) was synthesized, characterized with the desirable excellent hydrophilicity and thus was explored for the enrichment of N-linked glycopeptides utilizing the HILIC interaction between the glycopeptides and the hydrophilic UIO-PBA&FDP at a high level of ACN concentration. A total of 359 N-linked glycopeptides corresponding to 104 glycoproteins were identified from only 1 μL of digested human serum by the enrichment of UIO-PBA&FDP, which showed a superiorly high coverage of the identified glycopeptides. The dual hydrophilic functionalized UIO-PBA&FDP could be an efficient HILIC material for the enrichment of N-linked glycopeptides from complex biological samples.

Facile fabrication of hydrophilic covalent organic framework composites for highly selective enrichment of N-glycopeptides

The development of facilely synthetic materials acts an essential role in glycoproteome analysis, especially for the highly efficient enrichment of N-linked glycopeptides. In this work, a facile and timesaving route was introduced in which COFTP-TAPT served as a carrier and poly (ethylenimine) (PEI) and carrageenan (Carr) were successively coated on the surface via electrostatic interaction. The resultant COFTP-TAPT@PEI@Carr showed remarkable performance in glycopeptide enrichment with high sensitivity (2 fmol μL−1), high selectivity (1:800, molar ratio of human serum IgG to BSA digests), large loading capacity (300 mg g−1), satisfactory recovery (102.4 ± 6.0%) and reusability (at least eight times). Due to the brilliant hydrophilicity and electrostatic interactions between COFTP-TAPT@PEI@Carr and positively charged glycopeptides, the prepared materials could be applied in the identification and analysis in the human plasma of healthy subjects and patients with nasopharyngeal carcinoma. As a result, 113 N-glycopeptides with 141 glycosylation sites corresponding to 59 proteins and 144 N-glycopeptides with 177 glycosylation sites corresponding to 67 proteins were enriched from 2 μL plasma trypsin digests of the control groups and patients with nasopharyngeal carcinoma, respectively. 22 glycopeptides were identified only from the normal controls and 53 glycopeptides were detected only from the other set. The results demonstrated that this hydrophilic material was promising on a large scale and further N-glycoproteome research.

New hydrophilic material based on hydrogel polymer for the selective enrichment of intact glycopeptides from serum protein digests

The paper describes the preparation and characterization of a new HILIC material for the enrichment of N-linked glycopeptides. The material was prepared using 2-acrylamido-2-methyl-1-propanesulfonic acid as the monomer and ethylene glycol dimethacrylate as the cross-linker. The material was developed by a Box-Behnken experimental design, taking into consideration the amount of monomer-to-crosslinker ratio, the composition, and the amount of porogen mixture. By this approach, the property of the resulting polymer could be fine-tuned to modulate the hydrophilicity and porosity. As HILIC enrichment is mostly dependent on hydrophilic interactions, including H-bonding, the amount of swelling was expected to have an important function, therefore the optimization considered a monomer percent in the range of 20–80%, which implied very different water swelling capacities. After assessing the potential of this new polymer family on fetuin digests, the 17 materials resulting from the Box-Behnken experimental design were used for the enrichment of glycopeptides from serum protein digests. The materials displayed a superior performance over cotton HILIC enrichment, both in terms of the number of enriched N-linked glycopeptides and selectivity, providing up to 762 N-linked glycopeptides with 77% selectivity. The optimization indicated that a high amount of monomer significantly affected the number of enriched glycopeptides, which is also closely connected with the hydrogel nature of the resulting polymers. The results not only provide one additional HILIC material for the enrichment of glycopeptides but also pave the way for the use and development of hydrogel materials for the enrichment of N-linked glycopeptides.

Advances in hydrophilic metal-organic frameworks for N-linked glycopeptide enrichment.

The comprehensive profiling of glycoproteins is of great significance for the timely clinical diagnosis and therapy. However, inherent obstacles hamper their direct analysis from biological samples, and specific enrichment prior to analysis is indispensable. Among the various approaches for glycopeptide enrichment, hydrophilic interaction liquid chromatography (HILIC) has attracted special focus, especially for the development of novel hydrophilic materials, which is the key of HILIC. Metal-organic frameworks (MOFs) are a type of porous materials constructed from the self-assembly of metal and organic linkers. Advantages such as high surface area, flexible pore size, and easy modification render hydrophilic MOFs as ideal candidates for HILIC, which has inspired many studies over the past years. In this review, advances in hydrophilic MOFs for N-linked glycopeptide enrichment are summarized. According to the synthesis strategies, those materials are categorized into three classes, namely pristine MOFs, MOFs with chemical modifications, and MOFs-derived composite. In each categorization, the preparation and the function of different moieties are covered, as well as the enrichment performances of sensitivity, selectivity, and practical application. Finally, a summary and future perspective on the applications of hydrophilic MOFs for N-linked glycopeptide enrichment are briefly discussed. This review is expected to raise awareness of the properties of hydrophilic MOFs and offer some valuable information to further research in glycoproteomics.

One-Step Synthesis of Hydrophilic Nano-Floral Inter-Polymeric Material for Highly Selective Enrichment of N-Linked Glycopeptides

A novel nano-floral inter-polymeric material (λ-Carr-Ag-SA) was developed to effectively capture N-linked glycopeptides from biological samples. A simple one-step reduction of silver ions was involved in the synthesis of the material in which λ-carrageenan and sodium alginate, with abundant hydrophilic groups, were used as the reducing agent and stabilizer. The performance of λ-Carr-Ag-SA was evaluated experimentally by enriching glycopeptides from horse radish peroxidase (HRP) and human serum. All the results demonstrated that λ-Carr-Ag-SA had a great selectivity, low detection limit and a large binding capacity toward glycopeptides. The successful application exhibits great potential of λ-Carr-Ag-SA for further glycoproteome analysis.

Gold nanoparticle-glutathione-functionalized porous graphene oxide-based hydrophilic beads for the selective enrichment of N-linked glycopeptides.

A three-dimensional structured porous graphene oxide-polyethylenimine bead (pGP) is synthesized for immobilizing gold nanoparticles and modifying glutathione molecules (denoted as pGP/AuG). The pGP/AuG has open pore structure, honeycomb-like channels, and excellent hydrophilicity. By taking advantages of the porous structure, abundant binding sites, and multivalent interactions between glycopeptides and both glutathione molecules and free amino groups, the pGP/AuG is adopted to the selective enrichment of N-linked glycopeptides with low limit of detection (2fmol), high enrichment selectivity (1:500), binding capacity (333.3mg/g), recovery yield (91.3 ± 2.1%), and repeatability (< 6.0% RSD) using matrix-assisted laser desorption/ionization time of flight mass spectrometrydetection method. Furthermore, the practical applicability of pGP/AuG is evaluated, in which 209 N-glycosylated peptides corresponding to 128 N-glycosylated proteins are identified from 1μL human serum in three independent analysis procedures, suggesting the great potential for application in glycoproteome fields.Graphical abstract Schematic presentation of preparation for porous graphene oxide-based hydrophilic beads (pGP/AuG) with honeycomb-like microstructure. The pGP/AuG was successfully used for enriching and identifying glycopeptides from actual biological sample.

One-step synthesis of hydrophilic microspheres for highly selective enrichment of N-linked glycopeptides

A polyacrylamide-based hydrophilic microsphere with a lot of hydroxyl groups on surface (PAM-OH HMS) was prepared in one step. The synthetic process was simple reverse suspension polymerization without any chemical derivation or grafting steps. The properties of obtained HMS were characterized by scanning electron microscopy (SEM), static water contact angle measurement, and FT-IR. The abundant hydroxyl groups on the surface make the material highly good hydrophilic and thus it was utilized for N-glycopeptides enrichment. The enrichment efficiency of PAM-OH HMSs was demonstrated by capturing N-linked glycopeptides from tryptic digest of human immunoglobulin G (IgG). The detection sensitivity for N-glycopeptides identification by MALDI-TOF MS was as low as 10 fmol for tryptic digest of standard human IgG. The selectivity of the HMS towards N-glycopeptides had almost no decrease when the molar ratio of BSA tryptic digest to IgG tryptic digest was increased from 10:1 to 100:1. Moreover, in the LC-MS/MS analysis of real biological sample, a total of 344 unique N-glycosites in 598 unique N-glycopeptides from 172 N-glycoproteins were identified from 2 μL human serum after deglycosylated by PNGase F, and 825 intact N-glycopeptides with different types of glycoform were detected when directly analyzed the N-glycopeptides enriched by PAM-OH HMS. To show the potential of our material in solving real biological issues, N-glycopeptides in the serum from hepatocelluar carcinoma (HCC) patient and health control were enriched and quantified. All the experiments demonstrated that this polyacrylamide-based hydrophilic microsphere shows a great potential to enrich the low-abundance N-glycopeptides for glycoproteome analysis of real complicated biological samples.

A hydrophilic two-dimensional titanium-based metal-organic framework nanosheets for specific enrichment of glycopeptides

A hydrophilic two-dimensional titanium-based metal-organic framework (MOF) nanosheets were prepared and applied to improve the glycopeptides enrichment efficiency. Seventeen N-linked glycopeptides from horseradish peroxidase (HRP) tryptic digests were readily identified and the results showed highly improved signal intensities. As the molar ratio of the mixture of tryptic digests of HRP, glycoprotein (HRP), and nonglycoprotein (BSA) reached 1:800:800, good selectivity of the Ti-MOF nanosheets were still clearly observed. As to human serum samples, 41 glycoproteins and 66 glycopeptides corresponding to 68 N-glycosylation sites were finally detected, which showed the good ability of the nanosheets material to enrich N-linked glycopeptides from complex human serum samples. Moreover, the Ti-MOF nanosheets exhibited unique stability and reusability even after being regenerated and reused for five times. It also performed a high sensitivity (1 × 10−10 M HRP), a good enrichment capacity (250 mg/g) for glycopeptides enrichment and a high enrichment recovery of 78%, suggesting a great application prospect for the enrichment of N-linked glycopeptides and the analysis glycoproteomic research.

Glutathione-modified ordered mesoporous silicas for enrichment of N-linked glycopeptides by hydrophilic interaction chromatography

Hydrophilic interaction chromatography (HILIC), as one of the important methods for glycopeptides enrichment, has attracted the attention of more and more researchers due to the high efficiency in glycopeptides enrichment. Ordered mesoporous silica materials have been used in many fields, such as catalysis, separation and drug delivery, owing to their adjustable pore size, excellent mechanical properties and ease of preparation. In our case, a series of ordered mesoporous silicas with different pore sizes (3.7–16.7 nm) were prepared via sol-gel reaction employing F127 as the template and TEOS as the silica source. After modified with glutathione by photo-initiated thiol-ene reaction, these materials exhibited hydrophilicity and could be used as adsorbents of HILIC for capturing N-linked glycopeptides from IgG and serum protein tryptic digests. Up to 26 N-glycopeptides were identified from IgG digest, and 565 N-glycopeptides and 330 N-glycosylation sites, mapped to 159 glycoproteins, were identified from 2 μL human serum digest, indicating the great ability for enriching N-glycopeptides from complex biological samples.

Boronic acid functionalized MOFs as HILIC material for N-linked glycopeptide enrichment.

Highly specific enrichment of N-linked glycopeptides from complex biological samples is crucial prior to mass spectrometric analysis. In this work, a hydrophilic metal-organic framework composite is prepared by the growth of UiO-66-NH2 on graphene sheets, followed by its post-synthetic modification to attach boronic acid to form GO@UiO-66-PBA. The fabrication of graphene oxide-MOF composite results in enhanced surface area with improved thermal and chemical stability. The synthesized MOF nanocomposite is characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and BET. A crystalline structure with high porosity offering large surface area and good hydrophilicity of the nanocomposite assists as an enrichment tool in glycoproteomics. The GO@UiO-66-PBA nanocomposite selectively enriches N-linked glycopeptides from tryptic digests of horseradish peroxidase (HRP) and immunoglobulin (IgG). GO@UiO-66-PBA nanoparticles show a low detection limit (1 fmol) and good specificity (1:200), reusability and reproducibility for N-linked glycopeptide enrichment from IgG digest. The binding capacity of GO@UiO-66-PBA is 84mg/g for protein concentration, with a good recovery of 86.5%. A total of 372N-linked glycopeptides corresponding to different glycoproteins are identified from only 1μL of human serum digest. Thus, the presented research work can be an efficient separation platform for N-linked glycopeptide enrichment from complex samples, which can be extended to cost-effective routine analysis. Graphical abstract.

Facile synthesis of hydrophilic magnetic graphene nanocomposites via dopamine self-polymerization and Michael addition for selective enrichment of N-linked glycopeptides

The development of methods to effectively capture N-glycopeptides from the complex biological samples is crucial to N-glycoproteome profiling. Herein, the hydrophilic chitosan–functionalized magnetic graphene nanocomposites (denoted as Fe3O4-GO@PDA-Chitosan) were designed and synthesized via a simple two-step modification (dopamine self-polymerization and Michael addition). The Fe3O4-GO@PDA-Chitosan nanocomposites exhibited good performances with low detection limit (0.4 fmol·μL−1), good selectivity (mixture of bovine serum albumin and horseradish peroxidase tryptic digests at a molar ration of 10:1), good repeatability (4 times), high binding capacity (75 mg·g−1). Moreover, Fe3O4-GO@PDA-Chitosan nanocomposites were further utilized to selectively enrich glycopeptides from human renal mesangial cell (HRMC, 200 μg) tryptic digest, and 393 N-linked glycopeptides, representing 195 different glycoproteins and 458 glycosylation sites were identified. This study provides a feasible strategy for the surface functionalized novel materials for isolation and enrichment of N-glycopeptides.

Enrichment of N-Linked Glycopeptides and Their Identification by Complementary Fragmentation Techniques.

N-linked glycans are a ubiquitous posttranslational modification and are essential for correct protein folding in the endoplasmic reticulum of plants. However, this likely represents a narrow functional role for the diverse arrayof glycan structures currently associated with N-glycoproteins in plants. The identification of N-linked glycosylation sites and their structural characterization by mass spectrometry remains challenging due to their size, relative abundance, structural heterogeneity, and polarity. Current proteomic workflows are not optimized for the enrichment, identification and characterization of N-glycopeptides. Here we describe a detailed analytical procedure employing hydrophilic interaction chromatography enrichment, high-resolution tandem mass spectrometry employing complementary fragmentation techniques (higher-energy collisional dissociation and electron-transfer dissociation) and a data analytics workflow to produce an unbiased high confidence N-glycopeptide profile from plant samples.

Amino Acid-Functionalized Two-Dimensional Hollow Cobalt Sulfide Nanoleaves for the Highly Selective Enrichment of N-Linked Glycopeptides.

Leaf-like hollow cobalt sulfides with a sulfur-gold-cysteine (S-Au-Cys) structure on the surface have been synthesized for efficient N-glycopeptide enrichment. A two-dimensional (2D) zeolitic imidazolate framework with cobalt (ZIF-L-Co, L for leaf) was used as a self-sacrificed template. After sulfidation, the S-Au-Cys architecture was created on the surface of the leaf-like hollow cobalt sulfide to obtain a material denoted ZIF-L-Co-S-Au-Cys. Enrichment of glycopeptides from trypsin digests of immunoglobulin G (IgG) standard samples and of IgG isolated from real human plasma samples was accomplished via hydrophilic interaction liquid chromatography processes using ZIF-L-Co-S-Au-Cys. The good sensitivity and selectivity ensure the effectiveness and robustness of ZIF-L-Co-S-Au-Cys for sample preconcentration, which is comparable to a commercial HILIC product. This work provides an efficient way to produce transition metal sulfides with a low-dimensional morphology and provides a novel concept for material design for exploitation in sample preparation, especially in glycoproteomics.

Hydrophilic maltose-modified magnetic metal-organic framework for highly efficient enrichment of N-linked glycopeptides

Biomedical sciences, and in particular disease biomarker research, demand highly selective and efficient glycoproteins/peptides enrichment platforms. In this work, a facile strategy to prepare hydrophilic maltose-functionalized magnetic metal-organic framework loaded with Au nanoparticles (denoted as magMOF@Au-maltose) for highly efficient enrichment of N-linked glycopeptides. In brief, carboxyl-functional Fe3O4 nanospheres were firstly coated with a Zr-based MOF shell, the resulting MOF was then loaded with Au nanoparticles in situ and then modified with thiol-functional maltose via Au−S bonds to obtain magMOF@Au-maltose with core-shell structure. The physical property and adsorption of magMOF@Au-maltose to glycopeptides were investigated. The results showed magMOF@Au-maltose possessing the outstanding performance in glycopeptides enrichment with high selectivity (1:200, mass ratio of horseradish peroxidase to bovine serum albumin digest), a low limit of detection (10 fmol), a high recovery (over 83.3%), and a large binding capacity (83 μg•mg−1). The magMOF@Au-maltose nanocomposite can enrich 24 and 32 glycopeptides from tryptic HRP and human IgG digests, respectively. Moreover, the nanocomposite was applied to the selective enrichment of glycopeptides from the complex biological samples and a total of 123 unique N-glycosylation sites were identified from 113 glycopeptides in 1 μL of human serum, which were assigned to 46 different glycoproteins. These results showed the promising application of magMOF@Au-maltose in the detection and identification of low-abundance N-linked glycopeptides in complex biological samples.

Glutathione-Functionalized Magnetic Covalent Organic Framework Microspheres with Size Exclusion for Endogenous Glycopeptide Recognition in Human Saliva.

Endogenous glycopeptides have been confirmed to play a significant role in multifarious pathological and physiological processes. The low abundance of endogenous glycopeptides and abundant interferents (e.g., large-size proteins and heteropeptides) in complex biological matrices render the direct analysis of endogenous glycopeptides difficult. Reported here is a novel glutathione-functionalized magnetic covalent organic framework microsphere (denoted as MCNC@COF@GSH) endowed with size-exclusion effect and strong hydrophilicity for selective and efficient enrichment of N-linked glycopeptides. The as-prepared MCNC@COF@GSH microspheres possessed fast magnetic responsiveness, regular porosity, large surface areas, and good hydrophilicity, resulting in remarkable performances in N-linked glycopeptide enrichment with low detection limit (0.01 fmol μL-1), high selectivity (1:5000, human immunoglobulin G (IgG) digests to bovine serum albumin digests), excellent size-exclusion effect (IgG digests/IgG/bovine serum albumin (BSA), 1:500:500), and reusability (at least five times). More excitingly, 143 endogenous N-linked glycopeptides were clearly identified from 10 μL sample of human saliva treated with the MCNC@COF@GSH microspheres, which is the unprecedented high efficiency in endogenous N-linked glycopeptide enrichment from human saliva. In addition to providing a strategy for versatile functionalization of magnetic covalent organic frameworks (COFs), this study may be used to develop application of endogenous glycoproteome analysis.

Post-Synthetic Modification of Phenylboronic Acid-Functionalized Magnetic Covalent Organic Frameworks for Specific Enrichment of N-Linked Glycopeptides

Mass spectrometry (MS)-based in-depth glycoproteomics demands additional sample pretreatment to remove interference from nonglycoproteins and to enhance the precise identification of low-abundance ...

L-cysteine-modified metal-organic frameworks as multifunctional probes for efficient identification of N-linked glycopeptides and phosphopeptides in human crystalline lens

Highly selective enrichment of N-linked glycopeptides and phosphopeptides from complex biological samples is extremely important prior to mass spectrometry analysis due to their low abundance as well as numerous extrinsic interferences. In this work, l-cysteine (L-Cys)-modified multifunctional metal-organic frameworks denoted as Fe3O4@PDA@MIL-125@Au@L-Cys (mMIL-125@Au@L-Cys) were prepared by modifications step by step. By combining hydrophilic interaction chromatography (HILIC) with metal oxide affinity chromatography (MOAC), the as-prepared material was firstly utilized to identify N-linked glycopeptides and phosphopeptides from tryptic digests of horseradish peroxidase (HRP) and beta-casein (β-casein), respectively, with the help of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and exhibited outstanding sensitivity (0.1 fmol μL−1), great reusability (5 circles) and high selectivity (1: 100). Based on this, it was further applied into the enrichment of glycopeptides and phosphopeptides from tryptic digests of 100 μg human crystalline lens proteins. In the end, 81 N-linked glycopeptides corresponding to 35 glycoproteins and 175 phosphopeptides ascribed to 55 phosphorylated proteins were identified, respectively. The remarkable results were benefitted from the merits of improved hydrophilicity from L-Cys, strong affinity of TiO centers, numerous reaction sites on the large surface of MOFs and superparamagnetism from Fe3O4 cores. The design of mMIL-125@Au@L-Cys not only served as a multifunctional probe for efficient identification of N-linked glycopeptides and phosphopeptides in human crystalline lens, but also set a precedent for fabricating more MOFs with post-modifications for further proteomics research.

A facilely synthesized glutathione-functionalized silver nanoparticle-grafted covalent organic framework for rapid and highly efficient enrichment of N-linked glycopeptides.

The development of facilely synthetic materials for highly efficient enrichment of N-linked glycopeptides is essential in glycoproteome analysis. In this work, by utilizing the self-assembling of glutathione (GSH) on silver nanoparticles (Ag NPs), and the formation and dispersion of Ag NPs on a robust TpPa-1 substrate, a newly functionalized covalent organic framework (COF) called TpPa-1@Ag@GSH was synthesized via a simple two step post-synthetic modification. TpPa-1@Ag@GSH and intermediate products were confirmed and evaluated by nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, transmission electron microscopy, scanning electron microscopy-energy dispersive spectroscopy, Brunauer-Emmett-Teller and thermogravimetric analyses. Benefiting from the judicious selection of the substrate, the abundance of binding sites, relatively high affinity between GSH and N-linked glycopeptides, and the multivalent interactions between N-linked glycopeptides and unoccupied surfaces of Ag NPs, this porous material showed great performance in N-linked glycopeptide enrichment. By enriching N-linked glycopeptides in tryptic digests of human serum immunoglobulin G (human IgG) followed by mass spectrometry analysis, our method was proved to have good sensitivity (1 fmol), high selectivity (1 : 1500, human IgG to bovine serum albumin), high binding capacity (160 mg g-1, IgG/TpPa-1@Ag@GSH), ultra-fast capture ability (only 1 min incubation time), and good reusability (at least 5 times). It was also successfully applied to the enrichment of N-linked glycopeptides from complex biological samples. Our work improved the enrichment selectivity of COFs, reached the most rapid capture ability among off-column enrichment materials, and provided a very facile and easily popularized post-synthetic modification route for COFs in glycoproteome analysis.

Preparation of iminodiacetic acid functionalized silica capillary trap column for on-column selective enrichment of N-linked glycopeptides

In this work, SiO2-IDA microspheres were obtained via a previous method by grafting iminodiacetic acid (IDA) groups onto silica microspheres, then the SiO2-IDA microspheres were packed in capillary columns to prepare SiO2-IDA capillary trap columns for the first time. Based on the trap columns, a novel on-column enrichment method toward glycopeptides was developed as a sample pretreatment step prior to matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) or nano-LC-MS/MS analysis. The preparation procedures of the trap columns are well reproducible. Thanks to outstanding hydrophilicity brought by the grafted zwitterionic IDA groups, the trap columns showed excellent on-column enrichment performance toward glycopeptides by hydrophilic interaction liquid chromatography (HILIC). Experimental results showed that the on-column enrichment method could reach a low detection limit (50 fmol HRP digest) and a high selectivity (molar ratio of HRP digest and BSA digest 1:100). Finally, it was applied to on-column enrichment of glycopeptides from digests of human serum followed by nano-LC-MS/MS analysis. In total, 207 glycopeptides assigned to 78 glycoproteins were identified from 2 μL human serum.

Self-Assembling Glutamate-Functionalized Cyclodextrin Molecular Tube for Specific Enrichment of N-Linked Glycopeptides.

Cyclodextrin molecular tube (CDMT), a new comer of cyclodextrin family, possesses large and hydrophilic outer area and stable structure. Its development and applications remain highly desired, especially in the field of separation and enrichment. Herein, we developed a CDMT-based enrichment platform focusing on the specific capture of glycopeptides. To enhance the hydrophilicity of CDMT, it was functionalized with glutamate (glu). The prepared gluCDMT exhibited large hydrophilic surface, high stability, and good acidic/alkalic resistance. A solid monolithic support was employed to immobilize gluCDMT by a host-guest self-assembly synthetic strategy, which did not occupy the surface hydrophilic sites. The gluCDMT-based monolith exhibited high binding capacity (∼50 mg g-1), good ability to capture glycopeptides (23 HRP glycopeptides and 28 IgG glycopeptides), and high selectivity (horseradish peroxidase/bovine serum albumin = 1:10 000). Moreover, the developed platform was successfully applied to analyze glycopetides in acute myelogenous leukemia cell lysate and human serum samples.