Enhancing Extracellular Vesicle Analysis by Integration of Large-Volume Sample Stacking in Capillary Electrophoresis with Asymmetrical Flow Field-Flow Fractionation.

Extracellular vesicles (EVs) play important roles in cell-cell communication and pathological development. Cargo profiling for the EVs present in clinical specimens can provide valuable insights into their functions and help discover effective EV-based markers for diagnostic and therapeutic purposes. However, the highly abundant and complex matrix components pose significant challenges for specific identification of low-abundance EV cargos. Herein, we combine asymmetrical flow field-flow fractionation (AF4) with large-volume sample stacking and capillary electrophoresis (LVSS/CE), to attain EVs with high purity for downstream protein profiling. This hyphenated system first separates the EVs from the contamination of smaller serum proteins by AF4, and second resolves the EVs from the coeluted, nonvesicular matrix components by CE following LVSS. The optimal LVSS condition permits the injection of 10-fold more EVs into CE compared to the nonstacking condition without compromising separation resolution. Collection and downstream analysis of the highly pure EVs after CE separation were demonstrated in the present work. The high EV purity yields a much-improved labeling efficiency when detected by fluorescent antibodies compared to those collected from the one-dimension separation of AF4, and permits the identification of more EV-specific cargos by LC-MS/MS compared to those isolated by ultracentrifugation (UC), the exoEasy Maxi Kit, and AF4. Our results strongly support that AF4-LVSS/CE can improve EV isolation and cargo analysis, opening up new opportunities for understanding EV functions and their applications in the biomedical fields.

Short-Term HRV Detection and Human Fatigue State Analysis Based on Optical Fiber Sensing Technology.

Mental fatigue is a key cause of chronic diseases and traffic accidents, which is difficult to be quantitatively evaluated. In order to non-intrusively detect fatigue state, an optical fiber sensing system is proposed, which is non-invasive and does not require direct contact with skin. The fiber sensor was fabricated through phase mask exposure method and packaged by sensitivity-enhanced structure, which can suppress transverse force and increase signal amplitude by 5%. A fatigue-inducing experiment was carried out, and the heartbeat signals of 20 subjects under different fatigue states were collected by the proposed sensing system. A series of heart rate variability indicators were calculated from the sensing signals, and their statistical significance for fatigue was analyzed. The experiment results showed that the values of SDNN and LF/HF increased significantly with subjects' fatigue level. This study shows that the proposed fiber optic sensing system has practical value in fatigue state monitoring.

Chemoproteomic Approach toward Probing the Interactomes of Perfluoroalkyl Substances.

Poly- and perfluoroalkyl substances (PFASs) are widely used in industrial products and consumer goods. Due to their extremely recalcitrant nature and potential bioaccumulation and toxicity, exposure to PFASs may result in adverse health outcomes in humans and wildlife. In this study, we developed a chemoproteomic strategy, based on the use of isotope-coded desthiobiotin-perfluorooctanephosphonic acid (PFOPA) probe and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, to profile PFAS-binding proteins. Targeted proteins were labeled with the desthiobiotin-PFOPA probe, digested with trypsin, and the ensuing desthiobiotin-conjugated peptides were enriched with streptavidin beads for LC-MS/MS analysis. We were able to identify 469 putative PFOPA-binding proteins. By conducting competitive binding experiments using low (10 µM) and high (100 µM) concentrations of stable isotope-labeled PFOPA probes, we further identified 128 nonredundant peptides derived from 75 unique proteins that exhibit selective binding toward PFOPA. Additionally, we demonstrated that one of these proteins, fatty acid-binding protein 5 (FABP5), could interact directly with PFASs, including perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), and perfluorobutanesulfonic acid (PFBS). Furthermore, desthiobiotin-labeled lysine residues are located close to the fatty acid-binding pocket of FABP5, and the binding affinity varies with the structures of PFASs. Taken together, we developed a novel chemoproteomic method for interrogating the PFAS-interacting proteome. The identification of these proteins sets the stage for understanding the mechanisms through which exposure to PFASs confers adverse human health effects.

Effective Enrichment Strategy Using Boronic Acid-Functionalized Mesoporous Graphene-Silica Composites for Intact N- and O-Linked Glycopeptide Analysis in Human Serum.

The heterogeneity and low abundance of protein glycosylation present challenging barriers to the analysis of intact glycopeptides, which is key to comprehensively understanding the role of glycosylation in an organism. Efficient and specific enrichment of intact glycopeptides could help greatly with this problem. Here, we propose a new enrichment strategy using a boronic acid (BA)-functionalized mesoporous graphene-silica composite (denoted as GO@mSiO2-GLYMO-APB) for isolating intact glycopeptides from complex biological samples. The merits of this composite, including high surface area and synergistic effect from size exclusion functionality of mesoporous material, hydrophilic interaction of silica, and the reversible covalent binding with BA, enable the effective and specific enrichment of both intact N- and O-glycopeptides. The results from the enrichment performance of the strategy evaluated by standard glycoproteins and the application to global N- and O-glycosylation analyses in human serum indicate the robustness and potential of the strategy for intact glycopeptide analysis.

Puromycin-Modified Silica Microsphere-Based Nascent Proteomics Method for Rapid and Deep Nascent Proteome Profile.

Nascent proteome is crucial in directly revealing how the expression of a gene is regulated on a translation level. In the nascent protein identification, puromycin capture is one of the pivotal methods, but it is still facing the challenge in the deep profiling of nascent proteomes due to the low abundance of most nascent proteins. Here, we describe the synthesis of puromycin-modified silica microspheres (PMSs) as the sorbent of dispersive solid-phase microextraction and the establishment of the PMS-based nascent proteomics (PMSNP) method for efficient capture and analysis of nascent proteins. The modification efficiency of puromycin groups on silica microspheres reached 91.8% through the click reaction. After the optimization and simplification of PMSNP, more than 3500 and 3900 nascent proteins were rapidly identified in HeLa cells and mouse brains within 13.5 h, respectively. The PMSNP method was successfully applied to explore changes in the translation process in a biological stress model, namely, the lipopolysaccharide-stimulated HeLa cells. Biological functional analyses revealed the unique characters of the nascent proteomes and exhibited the superiority of the PMSNP in the identification of low abundance and secreted nascent proteins, thus demonstrating the sensitivity and immediacy of the PMSNP method.

Computational and Mass Spectrometry-Based Approach Identify Deleterious Non-Synonymous Single Nucleotide Polymorphisms (nsSNPs) in JMJD6.

The jumonji domain-containing protein 6 (JMJD6) gene catalyzes the arginine demethylation and lysine hydroxylation of histone and a growing list of its known substrate molecules, including p53 and U2AF65, suggesting a possible role in mRNA splicing and transcription in cancer progression. Mass spectrometry-based technology offers the opportunity to detect SNP variants accurately and effectively. In our study, we conducted a combined computational and filtration workflow to predict the nonsynonymous single nucleotide polymorphisms (nsSNPs) present in JMJD6, followed by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and validation. The computational approaches SIFT, PolyPhen-2, SNAP, I-Mutant 2.0, PhD-SNP, PANTHER, and SNPS&GO were integrated to screen out the predicted damaging/deleterious nsSNPs. Through the three-dimensional structure of JMJD6, H187R (rs1159480887) was selected as a candidate for validation. The validation experiments showed that the mutation of this nsSNP in JMJD6 obviously affected mRNA splicing or the transcription of downstream genes through the reduced lysyl-hydroxylase activity of its substrates, U2AF65 and p53, further indicating the accuracy of this prediction method. This research provides an effective computational workflow for researchers with an opportunity to select prominent deleterious nsSNPs and, thus, remains promising for examining the dysfunction of proteins.

Boronic acid-functionalized mesoporous magnetic particles with a hydrophilic surface for the multimodal enrichment of glycopeptides for glycoproteomics.

Glycosylation is an important mechanism of secondary protein processing. Large-scale profiling of glycopeptides released by proteolytic digestion of glycoproteins from biologic samples with complex compositions is limited due to their low abundance. Herein, we present a multimodal material based on boronic acid-modified mesoporous magnetic particles with a hydrophilic surface and enlarged pores around 10 nm. Multimodal enrichment successfully improved the enrichment specificity and efficiency of BMMP by synergistic interaction of hydrophilicity and boronic acid functional groups. The 10 nm pore size allows glycopeptides to enter the channel. Hydrophilic glycopeptides could be selectively enriched with an extremely low limit of detection (0.33 fmol per µL) and a high selectivity (1 : 100). From 2 µL of human serum, 328 unique glycopeptides from 101 glycoproteins were identified. A total of 33% of those glycoproteins overlapped with FDA-cleared blood serum biomarkers. It is expected that BMMP in the future can be used for large-scale biomedical glycoproteomics studies.

In Situ Synthesis of Magnetic Mesoporous Phenolic Resin for the Selective Enrichment of Glycopeptides.

Protein glycosylation is a significant participant in a mass of biological processes, which is a pivotal protein post-translational modification. Due to the low contents of glycopeptides compared with nonglycopeptides and the microheterogeneity of glycosylation sites, highly selective enrichment methods for the purification of glycopeptides are required for the comprehensive characterization of glycoproteomics. In this work, a type of magnetic mesoporous phenolic resin (MMP) was prepared using branched polyethylenimine (PEI) as a cross-linker from a homogeneous magnetic Fe3O4@SiO2 solution in a resorcinol/formaldehyde monomer aqueous system via an in situ emulsion polymerization procedure. The results showed that MMP exhibited good biocompatibility, a mesoporous structure, nitrogen-containing functionality, excellent hydrophilicity, and solvent resistance by using multiple characterization methods. By taking advantage of the interaction between hydrophilic groups on the MMP and glycan components on the glycopeptides, the acquired MMP was utilized to the selective capture of N-glycopeptides (human IgG or HRP tryptic digests/BSA proteins = 1:50), good recovery yield (70.18-97.23%), superior binding capacity (400 mg g-1), and excellent reproducibility. Based on the outstanding performance in standard glycoproteins tryptic digests enrichment, MMP was further used to capture N-glycopeptides from tryptic digests of human serum. A total of 15 unique N-glycopeptides were identified from an ultralow sample volume (0.025 µL) of human serum. Overall, we identified 356 unique N-glycopeptides corresponding to 119 glycoproteins from human serum (0.35 µL) in the overlap of three replicate analyses. All the results have demonstrated that MMP has great potential in large-scale N-glycoproteomics research.

Facile preparation of core-shell magnetic metal-organic framework nanospheres for the selective enrichment of endogenous peptides.

Facile preparation of core-shell magnetic metal-organic framework nanospheres by a layer-by-layer approach is presented. The nanospheres have high surface area (285.89 cm(2) g(-1)), large pore volume (0.18 cm(3) g(-1)), two kinds of mesopores (2.50 and 4.72 nm), excellent magnetic responsivity (55.65 emu g(-1)), structural stability, and good dispersibility. The combination of porosity, hydrophobicity, and uniform magnetism was exploited for effective enrichment of peptides with simultaneous exclusion of high molecular weight proteins. The nanospheres were successfully applied in the selective enrichment of endogenous peptides in human serum.

Magnesium Magic: State-of-the-Art Nanocrystalline Materials Paving the Way for Hydrogen Storage.

Hydrogen has been regarded as a promising alternative to traditional fossil fuels, presenting itself as a viable and environmentally friendly energy choice. The design and fabrication of highly efficient hydrogen storage materials is crucial to the wide utilization of hydrogen-based technologies. Magnesium-based nanocrystalline materials have received significant interest in the field of hydrogen storage due to their remarkable hydrogen storage capabilities and release efficiency. This review emphasizes on the most useful techniques including vapor deposition, sol-gel synthesis, electrochemical deposition, magnetron sputtering, and template-assisted approaches used for the fabrication of Magnesium-based nanocrystalline hydrogen storage materials (Mg-NHSMs), stressing their advantages, limitations, and recent advancements. These cutting-edge techniques demonstrate their significance in offering useful insights into the performance of Mg-NHSMs. Further, this review describes various applications of Mg-NHSMs. In addition, this review highlights the conclusion and future perspectives on the improvement of magnesium based nanocrystalline materials for efficient hydrogen storage.

Human Post-Translational SUMOylation Modification of SARS-CoV-2 Nucleocapsid Protein Enhances Its Interaction Affinity with Itself and Plays a Critical Role in Its Nuclear Translocation.

Viruses, such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infect hosts and take advantage of host cellular machinery for genome replication and new virion production. Identifying and elucidating host pathways for viral infection is critical for understanding the development of the viral life cycle and novel therapeutics. The SARS-CoV-2 N protein is critical for viral RNA (vRNA) genome packaging in new virion formation. Using our quantitative Förster energy transfer/Mass spectrometry (qFRET/MS) coupled method and immunofluorescence imaging, we identified three SUMOylation sites of the SARS-CoV-2 N protein. We found that (1) Small Ubiquitin-like modifier (SUMO) modification in Nucleocapsid (N) protein interaction affinity increased, leading to enhanced oligomerization of the N protein; (2) one of the identified SUMOylation sites, K65, is critical for its nuclear translocation. These results suggest that the host human SUMOylation pathway may be critical for N protein functions in viral replication and pathology in vivo. Thus, blocking essential host pathways could provide a novel strategy for future anti-viral therapeutics development, such as for SARS-CoV-2 and other viruses.

Optimized MALDI-TOF MS Strategy for Characterizing Polymers.

In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) plays an essential role in the analysis of polymers. To acquire a more reliable strategy for polymer profiling, we characterized four representative polymers including polyethylene glycol 6000, polyvinylpyrrolidone K12, polymer polyol KPOP-5040, and polyether polyol DL-4000. The preparation methods of these four polymer samples have been optimized from six aspects, including matrix, cationization reagent, solvent, mixing ratio of cationization reagent to polymer, mixing ratio of matrix to polymer, and laser intensity. After investigating the effects of seven commonly used matrices on the ionization efficiency of four polymers, trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene] malononitrile (DCTB) was found to be the only matrix suitable for the analysis of all the four polymers. Our experimental results suggested that different polymers showed a certain preference for different cationization reagents. For example, the polymer polyol KPOP-5040 was suitable for sodium iodide as the cationization reagent, while polyvinylpyrrolidone K12 was more suitable for silver trifluoroacetate (AgTFA). For the choice of solvent, tetrahydrofuran is a reagent with rapid evaporation and a wide range of dissolution which can achieve the best results for the analysis of four polymers. The optimized method was successfully applied to the identification of DSPE-PEG-NH2 with different polymerized degrees. This MALDI-TOF strategy potentially provided the supplementary function through the polymer's application in biomedical and visible probing.

Microarray investigation of glycan remodeling during macrophage polarization reveals α2,6 sialic acid as an anti-inflammatory indicator.

Glycosylation is a widely occurring posttranslational modification. Here, we applied a quick, convenient and high-throughput strategy (lectin array) to investigate the variation in glycans on different macrophage subtypes derived from THP-1 and RAW264.7 cells. For THP-1 cells, there were more significant differences in the glycan on M2 macrophages compared to the other two subtypes. In contrast, M1 macrophages exhibited more significant glycan remodeling than the other subtypes for the RAW264.7 cell line. The response of the lectins which recogonize the N-glycan and α2,6 sialic acid was higher during polarization into anti-inflammatory phase (THP-1 derived M2 subtypes), and lower in pro-inflammatory phase (RAW264.7 M1 subtypes). The regulation of several α2,6 sialyltransferase genes was coincident with the regulation of the α2,6 sialic acid on the two cell lines. The lectin response and glycosyltranferase gene expression confirmed that α2,6 sialic acid showed higher expression in the anti-inflammatory phase. This indicated that α2,6 sialic acid was a potential indicator for the anti-inflammatory response.

Precision N-Glycoproteomic Profiling of Murine Peritoneal Macrophages After Different Stimulations.

Macrophages are important immune cells that participate in both innate and adaptive immune responses, such as phagocytosis, recognition of molecular patterns, and activation of the immune response. In this study, murine peritoneal macrophages were isolated and then activated by LPS, HSV and VSV. Integrative proteomic and precision N-glycoproteomic profiling were conducted to assess the underlying macrophage activation. We identified a total of 587 glycoproteins, including 1239 glycopeptides, 526 monosaccharide components, and 8326 intact glycopeptides in glycoproteomics, as well as a total of 4496 proteins identified in proteomic analysis. These glycoproteins are widely involved in important biological processes, such as antigen presentation, cytokine production and glycosylation progression. Under the stimulation of the different pathogens, glycoproteins showed a dramatic change. We found that receptors in the Toll-like receptor pathway, such as Tlr2 and CD14, were increased under LPS and HSV stimulation. Glycosylation of those proteins was proven to influence their subcellular locations.

An ultrafast and highly efficient enrichment method for both N-Glycopeptides and N-Glycans by bacterial cellulose.

A powerful and fast glycopeptide/glycan enrichment method is critical for the efficiency and throughput of mass spectrometry (MS)-based glycoproteomic and glycomic analyses, especially for large-scale sample analysis. Here, we report an ultrafast and effective method for both intact N-glycopeptide and N-glycan enrichment and apply it to human serum samples. In this method, a natural hydrophilic material, bacterial cellulose (BC), was adopted and fully optimized for enrichment. This method offers the following advantages: (i) The enrichment material has natural hydrophilicity and is low-cost, biocompatible, biodegradable and easily accessible; (ii) the whole enrichment procedure is remarkably simple and fast. It takes only 10 min for intact glycopeptides/glycans to be easily purified from mixtures; (iii) the specificity of this method is over 94% for both glycan and glycopeptide enrichment; and (iv) the outstanding specificity of this technique enables high isolation efficiency for the enrichment of both intact glycopeptides and glycans. A total of 36 N-glycans and 31 N-glycopeptides were identified from human immunoglobulin G (IgG). The glycan and glycopeptide absorption capacity of BC was as high as 333 µg/mg and 250 µg/mg (IgG/BC) respectively. The selectivity for glycan and glycopeptide enrichment reached 1:100 (IgG/bovine serum albumin (BSA), molar ratio) and 1:200 (maltoheptaose (DP7)/BSA, molar ratio), respectively. Furthermore, a total of 159 N-glycans and 523 N-glycopeptides were identified in human serum by using this method. Overall, the BC-based enrichment method we present here provides an ultrafast and highly efficient method for the enrichment of both N-glycopeptides and N-glycans in complex samples and shows great potential in large-scale glycoproteomic and glycomic analyses.

Aperture-controllable nano-electrospray emitter and its application in cardiac proteome analysis.

The emitter clogging is the most common hardware failure of nano-electrospray ionization, to improve the durability and electrospray stability of fused silica emitters, we demonstrate a means of fabricating nano-electrospray emitters with controllable aperture size and gradually-narrowed channel on the tip. We simulated the fluid morphologies in the emitter channels by computational fluid dynamics and found more stable flow on aperture-controllable nano-electrospray emitter. Besides, we found the unstable flow sections of commercial emitters match the actual clogging sections very well, indicating the main cause of emitter clogging is unstable flow. We further tested the emitters by nano-LC-MS based proteome analysis. Compared with the commercial emitter, aperture-controllable nano-electrospray emitters promoted the total ion chromatogram intensity by 25%, the number of identified proteins by 6.58%, and the number of identified peptides by 7.87%. In total, 989 proteins were identified from 1 µg of extracted mouse cardiac proteins. After the optimization by using mouse samples, we analyzed clinical auricular dextral tissues from patients undergoing cardiac surgery and found 16 proteins related to atrial fibrillation. Overall, aperture-controllable nano-electrospray emitter exhibits better sensitivity and reproducibility in the application of nano-LC-MS cardiac proteome analysis.

Facile synthesis of layered mesoporous covalent organic polymers for highly selective enrichment of N-glycopeptides.

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.

Preparation of mesoporous carbon material derived from Metal-Organic Frameworks and its application in selective capture of endogenous peptides from human serum.

Highly selective and efficient extraction of endogenous peptides from complex biological systems plays an important role in proteomics. In this study, an ordered mesoporous magnetic material (denoted as CZIF) with micron size (1 µm) was facilely synthesized by direct pyrolyzing ZIF-67 under N2 atmosphere for efficient and selective enrichment of endogenous peptides. The CZIF keeps the morphology of the original ZIF-67 crystal with enlarged pore size of 3.67 nm and a specific surface area of 156.58 m2/g, and shows weak magnetism (18 emu/g). The loading buffer and eluting buffer were optimized to improve the sensitivity and selectivity of the CZIF. Benefiting from the highly graphitization of the CZIF, the limit detection of BSA digest as low as 0.2 fmol/µL was achieved, showing good enrichment sensitivity for peptides. The uniform mesoporous help to achieve high selectivity by excluding proteins (BSA tryptic digest/BSA = 1:1000). The CZIF can be repeatedly used (n ≥ 5) without further treatment. Moreover, 402 endogenous peptides were enriched from human serum by CZIF, which indicated that CZIF was a promising candidate for the extraction of endogenous peptides from complex biologicals.

Facile preparation of molybdenum (VI) oxide - Modified graphene oxide nanocomposite for specific enrichment of phosphopeptides.

To promote the development of phosphoproteome analysis in highly selective efficient tracing phosphorylated proteins or peptides, views of researches should not confined with intrinsic materials and their modification. New materials are supposed to be explored for phosphoproteome analysis. In this work, we first introduced Molybdenum (VI) oxide (MoO3) into phosphoproteome, loading on the graphene oxide (GO) nanosheets forming MoO3/GO nanocomposites by a simple two-step strategy. The GO nanosheets offered MoO3 a perfect stable platform for separation and concentration and MoO3 exhibited wonderful property in enriching phosphopeptides with highly selectivity and sensitivity on GO nanosheets. Specifically, the as-synthesized MoO3/GO nanocomposites exhibited excellent specificity (ß-casein: BSA=1:1000), high detection sensitivity (1 fmol/mL) and well recovery (91.13%) in enriching phosphopeptides by metal oxide affinity chromatography (MOAC). Moreover, the as-synthesized MoO3/GO nanocomposites provided effective enrichment of phosphopeptides from nonfat milk (a total of twelve phosphopeptides signals) and human serum (a total of four endogenous phosphopeptides signals), displaying great biological compatibility, which demonstrated that the MoO3/GO nanocomposites is a promising candidate in selectively identifying and determining low-abundance phosphorylated peptides in biological sample.

Facile preparation of 3-D floor-like ordered mesoporous carbon functionalized graphene composites and its application for selective enrichment of N-glycans from human serum.

Novel 3-D floor-like ordered mesoporous carbon functionalized graphene composites, FLOMC-GO, with high graphitized carbon contents were successfully synthesized using a soft template method. The one-pot sol-gel method was employed to prepare the silica soft template. Then, the sandwich-like composites were further combined together to form a 3-D structure through pre-carbonization and carbonization. During these procedures, the sulphonyl and sulfide bridges were formed by cross-linking processes to connect the phenyl rings. The prepared FLOMC-GO was confirmed to have a large pore volume (1.03cm3g-1), high BET surface area (544.99m2g-1) and well-ordered mesoporous structure with a narrow pore-size concentrated at 3.74nm. The content of carbon reached 80% and was highly graphitized. By taking advantage of the interactions between carbon and glycans, FLOMC-GO was utilized to enrich N-linked glycans from OVA and human serum. As expected, excellent size-exclusion was found during the enrichment of N-glycans released from OVA, and 25 N-linked glycans were identified. The intensity of glycans enriched by FLOMC-GO was 7 times to the result of active carbon, while the ratio of OVA digestion to BSA interfering proteins increased to 1:50. Additionally, 31 N-linked glycans in total were enriched from human serum. The relatively easy synthesis as well as ability to enrich N-linked glycans with high selectivity and efficiency makes FLOMC-GO a promising adsorbent material for the discovery of human serum biomarkers for disease diagnosis.