Enrichment Of Glycoproteins Research Articles

Simple and efficient enrichment and separation of glycoprotein by teamed boronate affinity magnetic carbon nanospheres

Protein glycosylation is a key regulatory mechanism that controls many physiological and pathological processes. Analyses of glycoproteins in biological samples require challenging enrichment steps in light of sample complexity and low glycoprotein abundance. Here, in order to develop an efficient method to enrich glycoproteins, a magnetic carbon nanospheres (MCs) rich in amino group and polyboric acid sites was designed and prepared. The teamed boronate affinity was formed by the self-assembled of amino group and boric acid, which can specifically capture glycoprotein containing cis-diol under neutral conditions. Firstly, a simple green hydrothermal method led to the production of a carbon nanospheres matrix with abundant surface hydroxyl and carboxyl moieties. Then, MCs were synthesized by solvothermal method. Subsequently, the MCs were modified with polyethyleneimine and boric acid in turn to obtain the proposed composite. The hydrophilicity of amino group and its hydrogen bond with glycoprotein, as well as the cyclization reaction of boric acid with cis-1,2-diol, are utilized to specifically bind to glycoprotein. The prepared magnetic nanospheres exhibited binding to glycoproteins with rapid kinetics and strong affinity. Adsorption equilibrium was reached within 40 min, and the adsorption capacities for the glycoproteins ovalbumin, transferrin and horseradish peroxidase were found to be 493, 346 and 326 mg/g, respectively, while the adsorption capacities for the non-glycoproteins bovine serum protein and lysozyme were 60 and 30 mg/g, respectively. This novel nanoparticle provides a simple and efficient method for the enrichment and separation of glycoproteins from biological samples.

On-line enrichment and separation of glycoprotein by capillary electrophoresis based on pH response coating column

A capillary column coated with 3-aminophenylboronic acid (APBA)-functionalized gold nanoparticles (AuNPs@APBA) was prepared via electrostatic self-assembly. The coated column exhibited anti-nonspecific adsorption of glycoproteins, enabling selective online enrichment during capillary electrophoresis (CE). First, gold nanoparticles (AuNPs) were synthesized using the sodium citrate reduction method. Then, APBA was self-assembled electrostatically on the surface of the AuNPs to obtain AuNPs@APBA. This nanomaterial was bonded to the inner wall of a capillary through ion adsorption to produce a AuNPs@APBA-coated capillary column. Glycoproteins were adsorbed via bond formation with boric acid groups under alkaline conditions (pH 8) to generate borate esters. Under acidic conditions (pH 3), the borate esters dissociated to release the glycoproteins, thereby achieving the selective online enrichment and separation of glycoproteins. The AuNPs and AuNPs@APBA were characterized using Fourier transform infrared spectroscopy, and their sizes and Zeta potentials were determined. In addition, the electroosmotic flow (EOF) of the AuNPs@APBA-coated capillary column was measured. The results showed that the surface of the AuNPs was successfully modified with APBA and that AuNPs@APBA was adsorbed on the inner wall of the capillary. The peak area of ovalbumin (OVA) on the AuNPs@APBA-coated column was 26.46 times higher than that on a bare column via conventional electrophoresis. In contrast, the peak area of bovine serum albumin (BSA) only increased by 8.47 times, indicating that the AuNPs@APBA coated column selectively enriched glycoproteins. Evaluation of the reproducibility and stability of this method revealed that the AuNPs@APBA coated capillary column could be used continually for 33-67 h. The relative standard deviations (RSDs) of the peak areas for intra-day (n=5) and inter-day (n=6) analyses were 2.2% and 3.0%, respectively. The developed method was successfully applied to enrich glycoproteins in a 1×106-fold diluted egg white sample. Glycoproteins were not detected using conventional electrophoresis on the bare column, whereas the AuNPs@APBA-coated capillary column effectively enriched and separated glycoproteins, resulting in a peak area of 10469 mAU·ms. Furthermore, the entire enrichment and separation process was completed within 3 min. This new online enrichment and separation method for glycoproteins has the advantages of low sample consumption, simple operation, and high separation efficiency.

Rapid, efficient and highly selective separation and enrichment of glycoprotein by surface-imprinted MOF nanoparticles loaded with high-density boric acid

Usually, glycoproteins are related to diseases or physiological processes; however, a serious challenge remains in realizing their highly specific recognition and enrichment via an imprinting strategy owing to their large molecular size, water solubility and high flexibility. Herein, we reported novel molecularly imprinted polymers based on a metal–organic framework (MOF) material, which achieved the specific enrichment of the target glycoprotein ovalbumin (OVA) by combining a boronate affinity strategy with directional surface imprinting technique. The MOF skeleton has a large specific surface area, thus resulting in a high loading density for the boric acid group (4.66 %) and a satisfactory adsorption capacity (482.56 mg/g). Boronate affinity strategy and the surface imprinting technique resulted in a high specificity of the target glycoprotein. Surface imprinting led to imprinting sites located on the shallow surface of nanoparticles, thus resulting in a rapid kinetic adsorption equilibrium (20 min). And the presence of an acyl group on phenylboronic acid led to the best enrichment effect under physiological pH (7.4). Furthermore, OVA in the real egg white was successfully analyzed, which confirmed the universality of this strategy.

One‐step enrichment and stepwise elution of glycoproteins and phosphoproteins by hydrophilic Ti4+‐immobilized dendrimer poly(glycidyl methacrylate) microparticles functionalized with polyethylenimine and phytic acid

Glycosylation and phosphorylation rank as paramount post‐translational modifications, and their analysis heavily relies on enrichment techniques. In this work, a facile approach was developed for the one‐step simultaneous enrichment and stepwise elution of glycoproteins and phosphoproteins. The core of this approach was the application of the novel titanium (IV) ion immobilized poly(glycidyl methacrylate) microparticles functionalized with dendrimer polyethylenimine and phytic acid. The microparticles possessed dual enrichment capabilities due to their abundant titanium ions and hydroxyl groups on the surface. They demonstrate rapid adsorption equilibrium (within 30 min) and exceptional adsorption capacity for β‐casein (1107.7 mg/g) and horseradish peroxidase (438.6 mg/g), surpassing that of bovine serum albumin (91.7 mg/g). Furthermore, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was conducted to validate the enrichment capability. Experimental results across various biological samples, including standard protein mixtures, non‐fat milk, and human serum, demonstrated the remarkable ability of these microparticles to enrich low‐abundance glycoproteins and phosphoproteins from biological samples.

Preparation, characterization and application of boronic acid functionalized porous polymer for glycoproteins enrichment from biological samples

Preparation, characterization and application of boronic acid functionalized porous polymer for glycoproteins enrichment from biological samples

An enrichment system for glycoproteins using microfluidic paper-based analytical device with colorimetric detection

We introduce a facile method for glycoprotein enrichment, separation, and detection using microfluidic paper-based analytical devices (μPADs). The μPADs comprise two distinct components: the enrichment region (Chip I) and the detection section (Chip II). The reusable Chip I is fabricated by immobilizing 3-aminophenylboronic acid (APBA) onto a circular paper substrate, demonstrating exceptional glycoprotein affinity. In disposable Chip II, glycoproteins react with periodic acid, generating carbonyl compounds that produce a purple-colored product upon reacting with the Schiff reagent. Alterations in color intensity are captured using an ordinary cell phone and are subsequently analyzed using Photoshop software. Observations note a linear increase in average color intensities from 1.0 × 10−3 to 5.0 × 10−3 mol/L, with an R2 value of 0.9927. The relative standard deviations (RSDs) of inter-day (n = 5) and intra-day (n = 5) assessments stand at 1.5 % and 1.4 %, respectively. Our proposed approach can effectively enrich an ovalbumin (OVA) solution with concentrations as low as 1.0 × 10−11 mol/L, resulting in enrichment rates of 107 to 108 times. The interference deviations of five substances relative to OVA lie within the range of − 2.0 % to + 2.0 %. The methodology successfully enriches glycoproteins from egg white samples diluted by a factor of 1 × 106, demonstrating that the proposed method is remarkably suitable for enriching glycoproteins from authentic samples.

Electrospun Fiber Membrane with the Dual Affinity of Chelation and Covalent Interactions for the Efficient Enrichment of Glycoproteins.

As important biomarkers of many diseases, glycoproteins are of great significance to biomedical science. It is essential to develop efficient glycoprotein enrichment platforms and investigate their adsorption mechanism. In this work, a conspicuous enrichment strategy for glycoproteins was developed by using an electrospun fiber membrane wrapped with polydopamine (PDA) and modified with 3-aminophenylboronic acid and nickel ions, named PAN/DA@PDA@APBA/Ni. The enrichment characteristics of PAN/DA@PDA@APBA/Ni toward glycoproteins were explored through adsorption behavior. Thanks to the existence of two sites of interaction (metal ion chelation and boronate affinity), PAN/DA@PDA@APBA/Ni exhibited significant enrichment capacity for glycoproteins, ovalbumin (604.6 mg/g), and human immunoglobulin G (331.0 mg/g). The adsorption kinetic results of glycoprotein ovalbumin on PAN/DA@PDA@APBA/Ni conform to the pseudo-first-order kinetic model in the first adsorption stage, while the second half adsorption stage is more in line with the pseudo-second-order kinetic model. Moreover, the physical characteristics of PAN/DA@PDA@APBA/Ni and subsequent adsorption experiments on electrospun fiber modified with only phenylboronic acid or nickel ions both confirmed two sites of interaction (metal ion chelation and boronate affinity, respectively). Furthermore, a stepwise elution method with dual-affinity interaction was designed and successfully applied to enrich glycoproteins in real biological samples. This work provides an idea for sample pretreatment, especially for the design of dual-affinity materials in glycoproteins enrichment.

Precursor carboxy-silica for functionalization with interactive ligands. III. Carbodiimide assisted preparation of immobilized lectin stationary phases for high performance lectin affinity chromatography of sub-glycoproteomics from cancer and disease free human sera

In this study, a precursor carboxy-silica support was demonstrated in the immobilization of two different lectins, namely concanavalin A (Con A) and wheat germ agglutinin (WGA) for use in high performance lectin affinity chromatography (LAC) for the selective capturing and enrichment of glycoproteins from healthy/disease free and cancer human sera. The lectin columns thus obtained (i.e., Con A- and WGA-columns) showed no nonspecific interactions toward some chosen standard glycoproteins and non-glycoproteins. Both columns were shown in sub-glycoproteomics enrichment from human sera including disease free and adenocarcinoma cancer sera. The collected fractions were subjected to LC-MS/MS for identification of the captured glycoproteins, whereby the total number of identified proteins using Con A column from disease-free and cancer sera were 164 and 188, respectively while 133 and 103 proteins were identified in the fractions captured by the WGA column from disease-free and cancer sera samples, respectively. Differentially expressed proteins (DEPs) between the disease free and cancer sera in both the Con A and WGA column fractions were identified via the plot of the abundance vs. the protein ratio whereby the binary logarithm of average intensities of cancer and disease free sera were plotted against the binary logarithm of cancer/disease free sera ratios. The proteins that exhibit log 2 (cancer/healthy) ratio values greater than +2 and less than −2 in both categories are considered as DEPs. Furthermore, for visualization of the data arrangement, Q-Q scatterplot were also used whereby the binary logarithm of cancer serum was plotted against the binary logarithm of disease-free serum for both Con A and WGA. For Con A column, 28 up-regulated and 10 down regulated proteins were identified with a total of 38 DEPs while only two being non-glycoproteins. Furthermore, the up-regulated, and down regulated proteins recorded for WGA column are 14 and 6, respectively, totaling 20 proteins including 3 non-glycoproteins. Some of the non-specific binding to lectin are most likely due to protein-protein interactions.

Identification of Core-Fucosylated Glycoproteins by Single-Step Truncation of N-Glycans.

Alpha-1,6 core fucosylation (CF) is a unique glycoform of N-glycans, and studies showed that CF modifications are involved in the occurrence and progression of various diseases and may provide potential disease biomarkers. Current strategies for the CF glycoproteome are often based on multistep enrichment of glycoproteins or glycopeptides and sequential cleavage with different glycosidases to truncate the N-glycans. Although the detection ability of low-abundance glycoproteins is improved, sample loss, high cost, and the time-consuming multistep operation also affect the reproducibility of results and the practicality of the method. Here we developed a single-step truncation (SST) strategy and evaluated its potential for the CF glycoproteome of human serum. The SST strategy has the advantages of fewer operational steps, lower cost, higher number of identifications, and better quantitative stability compared with previous approaches and provides an efficient solution for large-scale quantitative analysis of the CF glycoproteome. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Single-step truncation strategy for core fucosylation glycoproteome analysis in human serum Basic Protocol 2: Liquid chromatography-tandem mass spectrometry quantification of site-specific core fucosylation glycopeptides Alternate Protocol: Pretreatment of cellular samples of core fucosylation glycoproteome with single-step truncation strategy.

Construction of magnetic MOF@COF hybrid via covalent integration for simultaneous identification of glyco‑ and phospho-proteins in human urine

Urine plays a great role in reflecting the physiological and pathological conditions of kidney. Glyco- and phospho-proteins in urine are important indicator for diagnosing kidney diseases biomarkers, while their low concentration and extremely complex composition in urine samples make it difficult for direct analysis. Pretreatment of urine samples including acetone extraction or ultrafiltration concentration are often required before the enrichment procedure and subsequent analysis step. Herein, we developed a novel strategy to achieve “mega mergers” via covalent integration of two promising porous crystal materials (MOF@COF) on the magnetic core. The Ti-O groups in the MOF and high hydrophilicity of COF endow the resulting porous hybrid with high-efficiency simultaneous enrichment capacity of glyco- and phospho-proteins from urine samples without additional pretreatment steps. The enrichment mechanism and possible binding modes were validated by theoretical calculations. The synthesized magnetic MOF@COF successfully enriched glyco- and phospho-proteins from urine samples of patients with nephrotic syndrome and healthy controls, indicating that the protocol can be used to distinguish patients with nephrotic syndrome based on the amount of enriched glycoproteins and phosphoproteins. This work not only provides a new idea for the efficient enrichment of glyco- and phospho-proteins with porous hybrid material, but also broadens the research possibilities for biomarker discovery in urine-based liquid biopsy.

Recent advances of data‐independent acquisition mass spectrometry‐based proteomics

Recent advances of data‐independent acquisition mass spectrometry‐based proteomics

N-Linked Glycoproteome Analysis of Diosorea alata Tuber Shows Atypical Glycosylation and Indicates Central Role of Glycosylated Proteins in Tuber Maturation.

Glycosylation is an important post translational modification in plants. First analysis of N-linked glycosylated proteins of Dioscorea alata using Concanavalin A lectin affinity chromatography enrichment coupled with label free quantification is presented. In total, 114 enriched glycoproteins were detected. Signal P and sub-cellular localization showed 42.2% of proteins to be secretory. These included peroxidases, endochitinases, calreticulin, calnexin, thaumatins and lipid transfer proteins. Gene Ontology and MapMan analysis predicted the enriched glycoproteins to be involved in processes essential for tuber maturation namely: signal transduction, lignification, protein trafficking, endoplasmic reticulum quality control and cell wall remodeling. This was supported by biochemical validation of the essential glycoproteins. Interestingly, out of the two dioscorin isoforms, Dio B was the only N-glycosylated form. In silico analysis showed O-glycosylation sites in the other form, Dio A suggesting its similarity with sporamin, the storage protein of sweet potato. Absence of signal peptide in Dio B and the presence of non-canonical motif hints towards its atypical glycosylation. The analysis revealed that N-glycosylation of Dio B isoform maintains the activities associated with Dioscorin at maturity and provides an overview of protein N-glycosylation, enriching the glycoproteome database of plants especially tubers.

Abstract 1909: Enrichment and Analysis of Head and Neck Cancer Mucin-Domain Glycoproteins Reveals the Presence of Tumor-Derived Immunoglobulin A

Abstract 1909: Enrichment and Analysis of Head and Neck Cancer Mucin-Domain Glycoproteins Reveals the Presence of Tumor-Derived Immunoglobulin A

Strategies to Enrich, Identify, and Characterize Glycoproteome in Blood Plasma Using Liquid Chromatography High-Resolution Mass Spectrometry.

Blood and blood-derived components such as plasma and serum are considered as excellent resources that can be utilized to understand the biology of higher eukaryotic organisms including human beings. In research and clinical studies, blood plasma and serum are used to monitor health conditions, progression, and severity of diseases. Many of the disease-related studies utilize plasma and serum due to their disease relevance and accessibility as they can be collected from patients and healthy donors through minimally invasive approaches. Despite its significance, the unique proteome composition, complexity, wide dynamic range, and heterogeneity of the plasma proteins highlight critical factors that challenge the existing analytical technologies. Depletion of high abundant proteins is one among the accepted methods that can simplify the plasma proteome complexity; however, collateral loss of critical proteins should be anticipated. Selective protein enrichment seems to be a better alternative to depletion. Glycosylation of proteins is a dominant posttranslational modification known for its biological as well as diagnostic and therapeutic potential. Most of the reported therapeutic targets for diagnosis and monitoring are found to be glycosylated. In this chapter, a protocol for selective and reproducible enrichment of glycoproteins from blood plasma followed by identification through liquid chromatography high-resolution mass spectrometry has been documented.

Label-free designed nanomaterials enrichment and separation techniques for phosphoproteomics based on mass spectrometry

The surface chemical characteristics of nanomaterials have a substantial impact on the affinity probe used to enrich proteins and peptides for MALDI-MS analysis of a real human sample. Detecting phosphoproteins involved in signalling is always difficult, even with recent developments in mass spectrometry, because protein phosphorylation is often temporary from complicated mixtures. This review summarizes current research on the successful enrichment of various intriguing glycoproteins and glycol peptides using surface affinity materials with distinctive qualities such as low cost, excellent structural stability, diversity, and multifunction. As a consequence, this review will provide a quick overview of the scholars from various backgrounds who are working in this intriguing interdisciplinary field. Label-free cancer biomarkers and other diseases will benefit from future challenges.

Genome Wide Association Study with Imputed Whole Genome Sequence Data Identifies a 431 kb Risk Haplotype on CFA18 for Congenital Laryngeal Paralysis in Alaskan Sled Dogs.

Congenital laryngeal paralysis (CLP) is an inherited disorder that affects the ability of the dog to exercise and precludes it from functioning as a working sled dog. Though CLP is known to occur in Alaskan sled dogs (ASDs) since 1986, the genetic mutation underlying the disease has not been reported. Using a genome-wide association study (GWAS), we identified a 708 kb region on CFA 18 harboring 226 SNPs to be significantly associated with CLP. The significant SNPs explained 47.06% of the heritability of CLP. We narrowed the region to 431 kb through autozygosity mapping and found 18 of the 20 cases to be homozygous for the risk haplotype. Whole genome sequencing of two cases and a control ASD, and comparison with the genome of 657 dogs from various breeds, confirmed the homozygous status of the risk haplotype to be unique to the CLP cases. Most of the dogs that were homozygous for the risk allele had blue eyes. Gene annotation and a gene-based association study showed that the risk haplotype encompasses genes implicated in developmental and neurodegenerative disorders. Pathway analysis showed enrichment of glycoproteins and glycosaminoglycans biosynthesis, which play a key role in repairing damaged nerves. In conclusion, our results suggest an important role for the identified candidate region in CLP.

Boronate-immobilized cellulose nanofiber-reinforced cellulose microspheres for pH-dependent adsorption of glycoproteins

High strength and excellent selectivity are two important aspects of porous cellulose microspheres as adsorbents for protein separation. For this purpose, self-reinforced all-cellulose microspheres (SCMs) with high strength were fabricated using natural cellulose nanofibers (CNFs) as fillers and then immobilized via 3-aminophenylboronic acids as affinity ligands for selective enrichment of glycoproteins. In particular, the inherent stiffness of entrapped CNFs endowed SCMs with more inflexibility, because the stress can be efficiently transferred from the network of SCMs to the stiff CNFs during the separation process. Besides, SCMs, as an all-cellulose material with homogenous surface chemistry and pore structure characteristics, are more suitable as supports for adsorbents. Finally, the SCMs were immobilized with 3-aminophenylboronic acids (BA/EPI-SCMs) and tested their performance in affinity adsorption of glycoproteins. BA/EPI-SCMs showed fast separation, high adsorption amount, and excellent selectivity toward glycoproteins.

Construction of magnetic covalent organic frameworks functionalized by benzoboroxole for efficient enrichment of glycoproteins in the physiological environment

To date, the development of highly selective and efficient glycoproteins/peptides enrichment is still a challenge for mass spectrometry-based proteomic analysis. In this work, we reported a novel strategy to prepare a magnetic amide-linked covalent organic framework functionalized by benzoboroxole (denoted as Fe3O4@COF-ABB), which was then used as an adsorbent for the enrichment of glycoproteins. The physical and adsorption properties of Fe3O4@COF-ABB were fully investigated. The Fe3O4@COF-ABB presents a regular core-shell spherical structure, quick magnetic response performance, regular porosity, and multiple binding sites of phenylboronic acid. Taking advantage of these benefits, the synthesized magnetic composites exhibited a superior adsorption capacity (565.8 mg g−1) and high selectivity towards glycoprotein immunoglobulin G (IgG) under physiological state (pH 7.4). Additionally, the adsorbent Fe3O4@COF-ABB could be easily regenerated and reused 5 times with no reduction of enrichment performance. More importantly, the practical applications of Fe3O4@COF-ABB were further demonstrated by the selective adsorption of IgG from human serum. The present work represents a rational design of versatile functionalization of magnetic COFs, which demonstrates an avenue for the selective enrichment and analysis of IgG from real biological sample matrices.

N-linked glycoproteomic profiling in esophageal squamous cell carcinoma.

BACKGROUNDMass spectrometry-based proteomics and glycomics reveal post-translational modifications providing significant biological insights beyond the scope of genomic sequencing.AIMTo characterize the N-linked glycoproteomic profile in esophageal squamous cell carcinoma (ESCC) via two complementary approaches.METHODSUsing tandem multilectin affinity chromatography for enrichment of N-linked glycoproteins, we performed N-linked glycoproteomic profiling in ESCC tissues by two-dimensional gel electrophoresis (2-DE)-based and isobaric tags for relative and absolute quantification (iTRAQ) labeling-based mass spectrometry quantitation in parallel, followed by validation of candidate glycoprotein biomarkers by Western blot.RESULTS2-DE-based and iTRAQ labeling-based quantitation identified 24 and 402 differentially expressed N-linked glycoproteins, respectively, with 15 in common, demonstrating the outperformance of iTRAQ labeling-based quantitation over 2-DE and complementarity of these two approaches. Proteomaps showed the distinct compositions of functional categories between proteins and glycoproteins with differential expression associated with ESCC. Western blot analysis validated the up-regulation of total procathepsin D and high-mannose procathepsin D, and the down-regulation of total haptoglobin, high-mannose clusterin, and GlcNAc/sialic acid-containing fraction of 14-3-3ζ in ESCC tissues. The serum levels of glycosylated fractions of clusterin, proline-arginine-rich end leucine-rich repeat protein, and haptoglobin in patients with ESCC were remarkably higher than those in healthy controls.CONCLUSIONOur study provides insights into the aberrant N-linked glycoproteome associated with ESCC, which will be a valuable resource for future investigations.

Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade.

Advances in bioanalytical technology have accelerated the analysis of complex protein glycosylation, which is beneficial to understand glycosylation in drug discovery and disease diagnosis. Due to its biological uniqueness in the course of disease occurrence and development, disease-specific glycosylation requires quantitative characterization of protein glycosylation. We provide a comprehensive review of recent advances in glycosylation analysis, including workflows for glycoprotein digestion, glycopeptide separation and enrichment, and mass spectrometry sequencing. We specifically focus on different strategies for glycopeptide enrichment through physical interaction, chemical oxidation, or metabolic labeling of intact glycopeptides. Recent advances and challenges of O-glycosylation analysis are presented, and the development of improved enrichment methods combining different proteases to analyze O-glycosylation is also proposed.