Endogenous Glycopeptides Research Articles

Triazine-structured covalent organic framework nanosheets with inherent hydrophilicity for the highly efficient and selective enrichment of glycosylated peptides.

Protein glycosylation plays a crucial role in various biological processes and is related to various diseases. Highly specific enrichment of glycopeptides before mass spectrometry detection is crucial for comprehensive glycoproteomic analysis. However, it still remains a great challenge due to the absence of affinity materials with excellent enrichment efficiency. In this work, a triazine structure linked by a -NH- bond of two-dimensional (2-D) covalent organic framework (COF) nanosheets was synthesized as an affinity adsorbent for the selective capture of glycopeptides. In particular, by introducing hydrophilic monomers via a bottom-up approach, the 2-D COF (denoted as NENP-1) nanosheets were provided with abundant amino groups and inherent hydrophilicity. Owing to the specific surface area and excessive accessible sites for hydrophilicity, the resulting NENP-1 nanosheets exhibited an outstanding glycopeptide enrichment efficiency from standard samples with a superior detection sensitivity (1 × 10-10 M), good enrichment selectivity (1 : 800, HRP tryptic digest to BSA protein), excellent binding capacity (100 mg g-1), great reusability, and recovery (60.2%). Furthermore, using the NENP-1 nanosheet adsorbent, twenty-four endogenous glycopeptides in the serum of patients with gastric cancer were successfully identified by LC-MS/MS technology, which illustrates a promising prospective of hydrophilic COF nanosheets in glycoproteomics research.

Super-hydrophilic sulfonate-modified covalent organic framework nanosheets for efficient separation and enrichment of glycopeptides

Highly efficient extraction of glycopeptides prior to mass spectrometry detection is extremely crucial for glycoproteomic research, especially in disease biomarker research. Reported here is the first time by applying two-dimensional (2D) covalent organic framework (COFs) nanosheets for highly efficient enrichment of glycopeptides. Particularly, by incorporating hydrophilic monomers through a bottom-up strategy, the 2D COF nanosheets (denoted as NUS-9) displayed an ultra-high graft density of sulfonic groups and super-hydrophilicity. In addition, because of the large surface area, low steric hindrance, high chemical stability, and abundant accessibility sites of 2D COF nanosheets, NUS-9 exhibited remarkable efficiency for glycopeptide enrichment, involving excellent detection sensitivity (0.01 fmol μL−1), outstanding enrichment capability, and good enrichment selectivity (1:1500, horseradish peroxidase (HRP) tryptic digest to bovine serum albumin (BSA) tryptic digest), and recovery (92.2 ± 2.0%). Moreover, the NUS-9 was able to unambiguously detect 631 endogenous glycopeptides from human saliva, demonstrating an unparalleled high efficiency in glycopeptide enrichment. Gene ontology analyses of proteins from human saliva enriched by NUS-9 demonstrated its potential for comprehensive glycoproteome analysis.

Inherently hydrophilic mesoporous channel coupled with metal oxide for fishing endogenous salivary glycopeptides and phosphopeptides

Both glycosylation and phosphorylation exert crucial rule in multitudinous biological processes. For in-depth profiling of glycosylation and phosphorylation, a magnetic metal oxide is effectively coupled with inherently hydrophilic mesoporous channels (denoted as Fe3O4@TiO2@mSiO2-TSG). Based on the mechanism of hydrophilic interaction liquid chromatography (HILIC) and metal oxide affinity chromatography (MOAC), the Fe3O4@TiO2@mSiO2-TSG nanomaterial shows high capacity for simultaneously enriching glycopeptides and phosphopeptides. With human saliva collected in successive four days as practical biological sample, endogenous glycopeptides and phosphopeptides are efficiently enriched. Further gene ontology analysis reveals that the identified endogenous glycopeptides and phosphopeptides participate in diverse molecular functions and biological processes. This strategy is anticipated to promote variation analysis of salivary post-translational modifications.

Post-synthesis of boric acid-functionalized magnetic covalent organic framework as an affinity probe for the enrichment of N-glycopeptides.

A novel type of boric acid-functionalized magnetic covalent organic framework (mCOF) with polyethyleneimine (PEI) as a linker (denoted as mCOF@PEI@B(OH)2) has beenprepared through a post-synthesis strategy, which points out an achievable path for the construction of boronic acid-functionalized COFs. Based on the boric acid chemistry, the obtained core-shell structured mCOF@PEI@B(OH)2 can selectively isolate glycopeptides through the modified boronic acid groups. The mCOF@PEI@B(OH)2 exhibits excellent performance with good reusability (ten cycles), low detection limit (0.5 fmol·μL-1), size-exclusion effect, and relatively high loading capacity (80μg·mg-1), recovery yield (94.9 ± 2.8%), and selectivity (HRP digests:BSA digests = 1:500). Detection is done bymatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, 37 endogenous glycopeptides are captured from human saliva with mCOF@PEI@B(OH)2, providing effective proofs for its capability to capture low-abundance glycopeptides from actual biological samples.

Post-synthesis modification of covalent organic frameworks for ultrahigh enrichment of low-abundance glycopeptides from human saliva and serum

In this study, a novel type of covalent organic framework (COF) material rich in boronic acid sites was prepared through post-synthesis modification (TbBD@PEI@Au@4-MPBA). The surface of COF material had abundant carboxylic acid groups, which could bind a large amount of polyethyleneimine (PEI) through electrostatic interaction. At the same time, the amino groups on the PEI can be grafted with Au nanoparticles (Au NPs) in situ, and then 4-mercaptophenylboronic acid (4-MPBA) was modified by the reaction of Au and sulfhydryl groups. The massive grafting of boronic acid groups made the material's enrichment effect on glycopeptides expected. The results of experiments indicated that the composite material has high sensitivity (5 amol μL−1) and selectivity (1:1000). In addition, the material has outstanding stability and reusability, with a load capacity of about 100 mg g−1 and a recovery of 99.3 ± 2.2%. What's more, after enriched by TbBD@PEI@Au@4-MPBA, 56 endogenous glycopeptides from fresh human saliva were detected by MALDI-TOF MS, 56 unique glycopeptides corresponding to 31 glycoproteins from human saliva and 513 unique glycopeptides corresponding to 208 glycoproteins from serum of throat cancer patient were detected by nano-LC-MS/MS, respectively, which was expected to be applied to glycoproteomics research.

One-step fabrication of strongly hydrophilic mesoporous silica for comprehensive analysis of serum glycopeptidome

Glycopeptidome represents reliable predictors of physiological and pathological status. Obstructions mainly including low abundance of endogenous glycopeptides and varied interference necessitate glycopeptide enrichment prior to MS analysis. Inspired by the prevalence of hydrophilic interaction chromatography for glycopeptide enrichment, a novel magnetic mesoporous silica nanomaterial (Fe3O4@mSiO2-TSG) with strongly hydrophilic property was developed through a one-pot method. In this work, the gluconamide-containing organosilane is innovatively proposed to directly serve as the strongly hydrophilic silica source for fabrication of hydrophilic mesoporous silica nanomaterial for glycopeptidomics research. Apart from excellent hydrophilicity, Fe3O4@mSiO2-TSG also was equipped with large specific surface area, ordered mesopore channels and great magnetic responsiveness. With all the advantages, Fe3O4@mSiO2-TSG displayed remarkable size-exclusion effect and considerable reusability. Moreover, combined with nano-LC-MS/MS, the glycopeptidome of serum from breast cancer patients was analyzed comprehensively, which showed noteworthy difference from healthy serum through gene ontology analysis, indicating great potential of the approach for glycopeptidomics research.

Bowl-like mesoporous polydopamine with size exclusion for highly selective recognition of endogenous glycopeptides

It has been confirmed that endogenous glycopeptide plays an important role in a variety of pathological and physiological processes. However, direct analysis of endogenous glycopeptide is still a great challenge owing to the low abundance of endogenous glycopeptides and the presence of a large number of interfering substances such as large-sized proteins and heteropeptides in complex biological sample. Herein, we reported a novel bowl-like mesoporous polydopamine nanoparticle modified by carrageenan (denoted as MPDA@PEI@CA) with strong hydrophilicity and size-exclusion effect for high specificity enrichment of endogenous glycopeptides. Thanks to the suitable pore channel structure as well as strong hydrophilic surface, the as-prepared MPDA@PEI@CA nanoparticles exhibited prominent performance in enrichment of N-linked glycopeptide with ultrahigh selectivity (1:5000 M ratio of horseradish peroxidase (HRP) digests/bovine serum albumin (BSA) digests), low detection limit (5 fmol μL−1), outstanding size-exclusion ability (1:1000 mass of HRP/BSA), and unique reusability (five times). 125 N-glycosylation sites of 134 glycopeptides from 65 glycoproteins were identified from 2 μL sample of human serum treated with the MPDA@PEI@CA nanoparticles, which manifested the ability to enrich endogenous N-linked glycopeptides from complex biological samples. These results indicated that the bowl-like MPDA@PEI@CA nanoparticles with novel structure prepared in this work had great potential for glycopeptidome analysis.

Melamine foam assisted in-tip packed amine-functionalized titanium metal–organic framework for the selective enrichment of endogenous glycopeptides

Endogenous glycopeptides are significantly important in diverse pathological and physiological systems, but their direct analysis is severely hampered by their low abundance and presence of interfering species in biological fluids. In this study, we synthesized the amine-functionalized titanium metal-organic framework (NH2-MIL-125(Ti)) by a simple hydrothermal method, characterized and used for glycopeptides enrichment. The designed separation media is highly hydrophilic and stable which is suitable for hydrophilic interaction chromatography (HILIC). To make the process smooth, simple, reliable, and robust, NH2-MIL-125(Ti) crystals were packed in pipette-tip using hydrophilic melamine foam, as supporting frit. Free amine groups, present in the structure imparted hydrophilicity and a unique pattern of porosity, contributing to the size exclusion effect that excluded the large-sized proteins up to 1:700 peptide to protein ratio. The prepared MOF particles possessed regular porosity, high surface area, good hydrophilicity, and offered an in-tip flow-based set-up enhanced the enrichment performance for N-linked glycopeptides. The affinity material showed a detection limit of 1 fmol.µL−1 and selectivity up to 1:1000 (HRP digest to BSA digest). Moreover, repeatability and reusability were evaluated up to five rounds of enrichment using the same affinity tip, and scanning electron microscopic images revealed no structural changes in the MOF crystals. Finally, the MOF packed in pipette tip was applied to selectively capture the N-linked endogenous glycopeptides from a healthy saliva sample and 64 unique endogenous glycopeptides were identified. These results demonstrated the excellent potential of NH2-MIL-125(Ti) based affinity tip for glycopeptides which can be used to enrich trace glycopeptide biomarkers from the biological fluids.

Profiling of Endogenously Intact N-Linked and O-Linked Glycopeptides from Human Serum Using an Integrated Platform

Endogenous glycopeptides in serum are an invaluable resource for biomarker discovery. Due to the low abundance and the poor fragmentation in tandem mass spectrometry, the identification of endogenously intact glycopeptides still faces many challenges. Herein, an integrated platform is fabricated for the identification of N-linked and O-linked endogenously intact glycopeptides. In this platform, the high-temperature acid denaturation, ultrafiltration, and hydrophilic interaction chromatography steps are combined together for the highly efficient extraction of the endogenously intact glycopeptides from a small amount of serum. Additionally, the twin-spectra scheme and in silico deglycosylation strategy were applied for the identification of N-linked and O-linked endogenous glycopeptides, respectively. In total, 223 intact N-glycopeptides and 51 intact O-glycopeptides are identified from only 40 μL of the human serum sample. This is the first study reporting the identification of endogenously intact N-linked and O-linked glycopeptide and is also the largest data set of endogenously intact glycopeptides reported so far. The distributions of glycans among peptides and proteins and cleavage sites on peptides are further analyzed to seek the regulation of endogenous glycosylation for disease mechanism. The developed strategy provides a novel platform for the disease biomarker discovery.

Construction of Magnetic Covalent Organic Frameworks with Inherent Hydrophilicity for Efficiently Enriching Endogenous Glycopeptides in Human Saliva.

In this work, a magnetic covalent organic framework (COF) with inherent hydrophilicity (denoted mCTpBD) was synthesized through interface deposition of a hydrophilic COF shell on amino group-functionalized magnetite particles via the reaction between a carboxyl group-containing monomer and benzidine. Thanks to the superior hydrophilicity, appropriate porous structure, and easy magnetic separation, the resulting mCTpBD exhibited excellent performance in conveniently enriching glycopeptides from standard samples with a high sensitivity of 0.5 fmol μL-1 and strong size-exclusion effect of up to 1:1000 (w/w). Furthermore, by using the mCTpBD adsorbent, endogenous glycopeptides in saliva of healthy people and patients with inflammatory bowel disease were successfully enriched and identified by the combined liquid chromatography-mass spectrometry/mass spectrometry technology, which indicates a promising prospective of core-shell magnetic composite microspheres with a hydrophilic COF shell in glycoproteomics research.

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.

High Anti-Interfering Profiling of Endogenous Glycopeptides for Human Plasma by the Dual-Hydrophilic Metal-Organic Framework.

Glycopeptidome profiling provides large-scale information about the glycosylation level of endogenous peptides, reflecting the dynamic processes of disease occurrences and developments. However, endogenous glycopeptides are usually submerged in complex fluids containing a wide variety of interference molecules, such as high concentration proteins, nonglycopeptides, and salts, which confounds attempts to identify glycopeptidome. Here, a dual-hydrophilic metal-organic framework is developed to selectively capture endogenous glycopeptides in complex biological fluid. The hydrophilic matrix material provides specific selectivity toward glycopeptides, while the deliberate surface regulation using hydrophilic species enhances its interaction with glycopeptides. This hydrophilic probe presents an extremely high performance in anti-interfering enrichment of glycopeptides from mimic complex samples, even when the molar ratio of immunoglobulin G versus bovine serum albumin was up to about 1:5000. More excitingly, in the practical application of glycopeptidome analysis, a total of 380 endogenous N-glycopeptides with 180 unique N-glycopeptide sites were identified from human plasma. This strategy is expected to broaden the application of dual-hydrophilic MOF-based materials, especially in dealing with the challenges of extremely complex biological samples.

Fabrication of hydrophilic multilayer magnetic probe for salivary glycopeptidome analysis

Variations in salivary components are closely associated with the predisposition and state of disease, the abnormal changes of salivary glycopeptidome are usually discovered as perilous singals of serious disease. Therefore, the monitoring and analyzing of salivary glycopeptidome are of even more overriding importance. In this work, a low-cost layer-by-layer assembly strategy was adopted to fabricate a hydrophilic multilayer magnetic probe (dubbed Mag-m-G6P) for salivary glycopeptidome analysis. The successful construction of multilayer structure not only guaranteed the good dispersal of probe by protecting magnetic core from itself aggregation tendency, but also endowed the probe with multiple advantages including the good hydrophilicity, uniform mesopore size and strong magnetic responsiveness, etc. As expected, with the optimized experimental conditions, the multifunctional probe showed high enrichemnt sensitivity, unbiased enrichment ability, excellent size-exclusion ability and reusability and so on in the process of standard sample analysis. At last, the Mag-m-G6P was successfully applied to salivary glycopeptidome analysis on further combination with LC–MS/MS analysis, a total of 53 endogenous glycopeptides were identified from human saliva.

Hydrophilic probe in mesoporous pore for selective enrichment of endogenous glycopeptides in biological samples

As one of the most important post-translational modifications (PTMs) of peptidome, glycopeptidome is closely related to serious disease, especially to cancer. In order to specifically discover and analyze glycopeptidome biomarkers for clinical diagnosis of cancer on early-stage, it is crucial to develop efficient technique to analyze low-abundance of endogenous glycopeptides in biological samples. In this report, a hydrophilic probe in mesoporous pore (denoted as Fe3O4@mSiO2@G6P) was designed and prepared. By taking advantage of the excellent hydrophilicity and size-exclusion ability, we applied Fe3O4@mSiO2@G6P to capture glycopeptides from both horseradish peroxidase (HRP) and immunoglobulin (IgG) digests successfully. Moreover, a total of 39 and 25 endogenous glycopeptides were identified from healthy saliva and gastric saliva, respectively, indicating the great potential of this probe for the exploration of glycopeptidome biomarkers.

Site-specific characterization of N-linked glycosylation in human urinary glycoproteins and endogenous glycopeptides.

Glycosylation is a very important post-translational modification involved in various cellular processes, such as cell adhesion, signal transduction and immune response. Urine is a rich source of glycoproteins and attractive biological fluid for biomarker discovery, owing to its availability, ease of collection, and correlation with pathophysiology of diseases. Although the urinary proteomics have been explored previously, the urinary glycoproteome characterization remains challenging requiring the development and optimization of analytical and bioinformatics methods for protein glycoprofiling. This study describes the high confident identification of 472 unique N-glycosylation sites covering 256 urinary glycoproteins. Besides, 202 unique N-glycosylation sites were identified in low molecular weight endogenous glycopeptides, which belong to 90 glycoproteins. Global site-specific characterization of the N-linked glycan heterogeneity was achieved by intact glycopeptide analysis, revealing 303 unique glycopeptides most of them displaying complex/hybrid glycans composed by sialic acid and fucose. These datasets consist in a valuable resource of glycoproteins and N-glycosylation sites found in healthy human urine that can be further explored in different disorders, in which the N-linked glycosylation may be aberrant.

Highly specific revelation of rat serum glycopeptidome by boronic acid-functionalized mesoporous silica

Although the specific profiling of endogenous glycopeptides in serum is highly inclined towards the discovery of disease biomarkers, studies on the endogenous glycopeptides (glycopeptidome) have never been conducted because of several factors. These factors include the high dynamic range of serum proteins, the inadequacy of traditional sample preparation techniques in proteomics for low-molecular-weight (LMW) proteins, and the relatively low abundances of glycopeptides. Boronic acid-functionalized mesoporous silica was synthesized in this study to overcome the limitations of the state-of-the-art methods for glycopeptidome research. The boronic acid-functionalized mesoporous silica exhibited excellent selectivity by analyzing glycopeptides in the mixture of glycopeptides/non-glycopeptides at molar ratio of 1:100, extreme sensitivity (the limit of detection was at the fmol level), good binding capacity (40mgg−1), as well as the high post-enrichment recovery of glycopeptides (up to 88.10%). The as-prepared material possessing both glycopeptide-suitable pore size and glycopeptide-specific selectivity has shown special capability for enriching the endogenous glycopeptides. Fifteen unique glycosylation sites mapped to 15 different endogenous glycopeptides were identified in rat serum. The established protocol revealed for the first time the rat serum glycopeptidome.

The cytoplasmic droplet of rat epididymal spermatozoa contains saccular elements with Golgi characteristics.

The cytoplasmic droplet of epididymal spermatozoa is a small localized outpouching of cytoplasm of the tail of unknown significance. EM revealed flattened saccular elements as the near exclusive membranous component of the droplet. Light and electron microscopic immunolabeling for Golgi/TGN markers showed these saccules to be reactive for antibodies to TGN38, protein affinity-purified alpha 2,6 sialyltransferase, and anti-human beta 1,4 galactosyltransferase. The saccules were isolated by subcellular fractionation and antibodies raised against this fraction immunolabeled the saccules of the droplet in situ as well as the Golgi region of somatic epithelial cells lining the epididymis. The isolated droplet fraction was enriched in galactosyltransferase and sialyltransferase activities, and endogenous glycosylation assays identified the modification of several endogenous glycopeptides. EM lectin staining in situ demonstrated galactose and N-acetyl galactosamine constituents in the saccules. Endocytic studies with cationic and anionic ferritin as well as HRP failed to identify the saccules as components of the endocytic apparatus. Epididymal spermatozoa were devoid of markers for the ER as well as the Golgi-associated coatamer protein beta-COP. It is therefore unlikely that the saccular elements of the droplet participate in vesicular protein transport. However, the identification of Golgi/TGN glycosylating activities in the saccules may be related to plasma membrane modifications which occur during epididymal sperm maturation.

Reconstitution of the Golgi apparatus after microinjection of rat liver Golgi fragments into Xenopus oocytes.

We have studied the reconstitution of the Golgi apparatus in vivo using an heterologous membrane transplant system. Endogenous glycopeptides of rat hepatic Golgi fragments were radiolabeled in vitro with [3H]sialic acid using detergent-free conditions. The Golgi fragments consisting of dispersed vesicles and tubules with intraluminal lipoprotein-like particles were then microinjected into Xenopus oocytes and their fate studied by light (LM) and electron microscope (EM) radioautography. 3 h after microinjection, radiolabel was observed by LM radioautography over yolk platelet-free cytoplasmic regions near the injection site. EM radioautography revealed label over Golgi stacked saccules containing the hepatic marker of intraluminal lipoprotein-like particles. At 14 h after injection, LM radioautographs revealed label in the superficial cortex of the oocytes between the yolk platelets and at the oocyte surface. EM radioautography identified the labeled structures as the stacked saccules of the Golgi apparatus, the oocyte cortical granules, and the plasmalemma, indicating that a proportion of microinjected material was transferred to the surface via the secretion pathway of the oocyte. The efficiency of transport was low, however, as biochemical studies failed to show extensive secretion of radiolabel into the extracellular medium by 14 h with approximately half the microinjected radiolabeled constituents degraded. Vinblastine (50 microM) administered to oocytes led to the formation of tubulin paracrystals. Although microinjected Golgi fragments were able to effect the formation of stacked saccules in vinblastine-treated oocytes, negligible transfer of heterologous material to the oocyte surface could be detected by radioautography. The data demonstrate that dispersed fragments of the rat liver Golgi complex (i.e., unstacked vesicles and tubules) reconstitute into stacked saccules when microinjected into Xenopus cytoplasm. After the formation of stacked saccules, reconstituted Golgi fragments transport constituents into a portion of the exocytic pathway of the host cell by a microtubule-regulated process.