Composition characterization of various viperidae snake venoms using MS-based proteomics N-glycoproteomics and N-glycomics.

Viperidae snake species is widely abundant and responsible for most envenomation cases in Turkey. The structural and compositional profiles of snake venom have been investigated to study the venom component variation across different species and to profile the venom biological activity variation against prey. In this context, we used proteomics, glycoproteomics and glycomics strategies to characterize the protein, glycoproteins and glycan structural and compositional profiles of various snake venoms in the Viperidae family. Moreover, we compared these profiles using the downstream bioinformatics and machine learning classification modules. The overall mass spectrometry profiles identified 144 different proteins, 36 glycoproteins and 78 distinct N-glycan structures varying in composition across the five venoms. A high amount of the characterized proteins belongs to the glycosylated protein family Trypsin-like serine protease (Tryp_SPc), Disintegrin (DISIN), and ADAM Cysteine-Rich (ACR). Most identified N-glycans have a complex chain carrying galactosylated N-glycans abundantly. The glycan composition data obtained from glycoproteomics aligns consistently with the findings from glycomics. The clustering and principal component analyses (PCA) illustrated the composition-based similarities and differences between each snake venom species' proteome, glycoproteome and glycan profiles. Specifically, the N-glycan profiles of M. xanthina (Mx) and V. a. ammodytes (Vaa) venoms were identical and difficult to differentiate; in contrast, their proteome profiles were distinct. Interestingly, the variety of the proteins across the species highlighted the impact of glycosylation on the diversity of the glycosylated protein families. This proposed high throughput approach provides accurate and comprehensive profiles of the composition and function of various Viperidae snake venoms.

Prediction of gastric cancer by machine learning integrated with mass spectrometry-based N-glycomics.

Early and accurate diagnosis of gastric cancer is vital for effective and targeted treatment. It is known that glycosylation profiles differ in the cancer tissue development process. This study aimed to profile the N-glycans in gastric cancer tissues to predict gastric cancer using machine learning algorithms. The (glyco-) proteins of formalin-fixed parafilm embedded (FFPE) gastric cancer and adjacent control tissues were extracted by chloroform/methanol extraction after the conventional deparaffinization step. The N-glycans were released and labeled with a 2-amino benzoic (2-AA) tag. The MALDI-MS analysis of the 2-AA labeled N-glycans was performed in negative ionization mode, and fifty-nine N-glycan structures were determined. The relative and analyte areas of the detected N-glycans were extracted from the obtained data. Statistical analyses identified significant expression levels of 14 different N-glycans in gastric cancer tissues. The data were separated based on the physical characteristics of N-glycans and used to test in machine-learning models. It was determined that the multilayer perceptron (MLP) was the most appropriate model with the highest sensitivity, specificity, accuracy, Matthews correlation coefficient, and f1 scores for each dataset. The highest accuracy score (96.0 ± 1.3) was obtained from the whole N-glycans relative area dataset, and the AUC value was determined as 0.98. It was concluded that gastric cancer tissues could be distinguished from adjacent control tissues with high accuracy using mass spectrometry-based N-glycomic data.

Utilizing linkage-specific ethyl-esterification approach to perform in-depth analysis of sialylated N-glycans present on milk whey glycoproteins.

Glycosylation of milk whey proteins, specifically the presence of sialic acid-containing glycan residues, causes functional changes in these proteins. This study aimed to analyze the N-glycome of milk whey glycoproteins from various milk sources using a linkage-specific ethyl esterification approach with MALDI-MS (matrix-assisted laser desorption/ionization-mass spectrometry). The results showed that the N-glycan profiles of bovine and buffalo whey mostly overlapped. Acetylated N-glycans were only detected in donkey milk whey at a rate of 16.06%. a2,6-linked N-Acetylneuraminic acid (a2,6-linked NeuAc, E) was found to be the predominant sialylation type in human milk whey (65.16%). The amount of a2,6-linked NeuAc in bovine, buffalo, goat, and donkey whey glycoproteomes was 42.33%, 44.16%, 39.00%, and 34.86%, respectively. The relative abundances of a2,6-linked N-Glycolylneuraminic acid (a2,6-linked NeuGc, Ge) in bovine, buffalo, goat, and donkey whey were 7.52%, 5.41%, 28.24%, and 17.31%, respectively. Goat whey exhibited the highest amount of a2,3-linked N-Glycolylneuraminic acid (a2,3-linked NeuGc, Gl, 8.62%), while bovine and donkey whey contained only 2.14% and 1.11%, respectively.

An integrated stage-tip-based glycomic and glycoproteomic approach for simple and rapid N-glycosylation profiling of glycoproteins.

Glycosylation is a post-translational modification that plays an active role in many cellular events. It also regulates many functions of proteins. Monoclonal antibody-derived drugs are used to treat many diseases, and glycosylation affects the activity of such drugs developed. On the other hand, N-glycans may change in certain diseases. Therefore, rapid and efficient bioanalytical methods are needed for N-glycosylation profiling. The study aimed to develop an integrated stage-tip application for the simple and rapid N-glycosylation profiling of glycoproteins. A fast and inexpensive N-glycosylation profiling was achieved by integrating all glycoproteomic and glycomic sample preparation steps into a stage-tip. The glycomic approach of the integrated stage-tip reduces the N-glycan profiling time from 2 days to approximately 2.5 hours. It also allows the profiling of immunoglobulin G (IgG) N-glycopeptides directly from human plasma. In addition, N-glycosylation profiling can be done in the developed method without sorbents, C18 or others, such as strong-cation exchange, at the glycopeptide level.

Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry.

Conjugated N-glycans are considered next-generation bioactive prebiotic compounds due to their selective stimulation of beneficial microbes. These compounds are glycosidically attached to proteins through N-acetylglucosamines via specific asparagine residue (AsN-X-Ser/Thr). Certain bacteria such as Bifidobacterium longum subspecies infantis (B. infantis) have been shown to be capable of utilizing conjugated N-glycans, owing to their specialized genomic abilities. B. infantis possess a unique enzyme, Endo-ß-N-acetylglucosaminidase (EndoBI-1), which cleaves all types of conjugated N-glycans from glycoproteins. In this study, recombinantly cloned EndoBI-1 enzyme activity was investigated using various immobilization methods: 1) adsorption, 2) entrapment-based alginate immobilization, 3) SulfoLink-, and 4) AminoLink-based covalent bonding immobilization techniques were compared to develop the optimum application of EndoBI-1 to food processes. The yield of enzyme immobilization and the activity of each immobilized enzyme by different approaches were investigated. The N-glycans released from lactoperoxidase (LPO) using different immobilized enzyme forms were characterized using MALDI-TOF mass spectrometry (MS). As expected, regardless of the techniques, the enzyme activity decreased after the immobilization methods. The enzyme activity of adsorption and entrapment-based alginate immobilization was found to be 71.55% ± 0.6 and 20.32% ± 3.18, respectively, whereas the activity of AminoLink- and SulfoLink-based covalent bonding immobilization was found to be 58.05 ± 1.98 and 47.49% ± 0.30 compared to the free form of the enzyme, respectively. However, extended incubation time recovery achieved activity similar to that of the free form. More importantly, each immobilization method resulted in the same glycan profile containing 11 different N-glycan structures from a model glycoprotein LPO based on MALDI-TOF MS analysis. The glycan data analysis suggests that immobilization of EndoBI-1 is not affecting the enzyme specificity, which enables full glycan release without a limitation. Hence, different immobilization methods investigated in this study can be chosen for effective enzyme immobilization to obtain bioactive glycans. These findings highlight that further optimization of these methods can be a promising approach for future processing scale-up and commercialization of EndoBI-1 and similar enzymes.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release.

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, including protein folding, stability, enzymatic protection, and biological recognition. N-glycans attached to glycoproteins (such as lactoferrin, lactadherin, and immunoglobulins) cannot be digested by the host and reach the large intestine, where they are consumed by certain beneficial microbes. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate the gut microbiome's beneficial microorganisms. However, the isolation of these new classes of prebiotics requires novel enzymes. Here, we describe the recombinant production of novel glycosidases from different Bifidobacteria strains (isolated from infants, rabbits, chicken, and bumblebee) for improved N-glycan isolation from glycoproteins. The method presented in this study includes the following steps: molecular cloning of Bifidobacterial genes by an in vivo recombinational cloning strategy, control of transformation success, protein induction, and protein purification.

Potential Applications of Endo-ß-N-Acetylglucosaminidases From Bifidobacterium longum Subspecies infantis in Designing Value-Added, Next-Generation Infant Formulas.

Human milk is the optimal source of infant nutrition. Among many other health benefits, human milk can stimulate the development of a Bifidobacterium-rich microbiome through human milk oligosaccharides (HMOs). In recent years, the development of novel formulas has placed particular focus on incorporating some of the beneficial functional properties of human milk. These include adding specific glycans aimed to selectively stimulate the growth of Bifidobacterium. However, the bifidogenicity of human milk remains unparalleled. Dietary N-glycans are carbohydrate structures conjugated to a wide variety of glycoproteins. These glycans have a remarkable structural similarity to HMOs and, when released, show a strong bifidogenic effect. This review discusses the biocatalytic potential of the endo-ß-N-acetylglucosaminidase enzyme (EndoBI-1) from Bifidobacterium longum subspecies infantis (B. infantis), in releasing N-glycans inherently present in infant formula as means to increase the bifidogenicity of infant formula. Finally, the potential implications for protein deglycosylation with EndoBI-1 in the development of value added, next-generation formulas are discussed from a technical perspective.

Differential N-glycosylation profiling of formalin-fixed paraffin-embedded (FFPE) invasive ductal carcinoma tissues using MALDI-TOF-MS.

Invasive ductal carcinoma (IDC) is the most common type of breast cancer. As dynamic changes of the glycome are closely associated with complex diseases, they have become a focal point of cancer research involving predictive and prognostic markers. Formalin-fixed paraffin-embedded (FFPE) clinical specimens are representative of the tumor environment and are thus utilized in studies on cancer related research and biomarker discovery. Further studies on differential N-glycosylation profiling of IDC cancer tissues are necessary in order to understand the biological role of glycans in cancer and to evaluate their predictive ability. In this study, matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS)-based analyses were conducted for determining differential N-glycosylation patterns of IDC. Two different derivatization methods, namely, 2-aminobenzoic acid (2-AA) labeling and linkage-specific sialic acid esterification, were used for the analysis of N-glycans. Forty-seven 2-AA labeled and fifty ethyl esterified N-glycans were identified by MALDI-MS. In statistical analyses conducted for 2-AA-labeled N-glycans, the relative amounts of 32 N-glycans and prevalence of 15 N-glycan traits showed significant (p < 0.05) differences between cancer and normal tissues; and in such analyses for the ethyl-esterified N-glycans, the relative amounts of 27 N-glycans and prevalence of 17 N-glycan traits showed significant (p < 0.05) differences between them. It was found that mainly high mannose N-glycans, including H5N2, H6N2, and H7N2, and two fucosylated compositions (H3N3F1 and H5N5F1) showed strong discrimination between IDC and controls. In addition, compared with the controls, high mannose N-glycans were observed to be up-regulated in IDC whereas bisecting N-glycans were down-regulated.

Hydrophilic spacer-arm containing magnetic nanoparticles for immobilization of proteinase K: Employment for speciation of proteins for mass spectrometry-based analysis.

Proteinase K (ProK) is used for the degradation of proteins in cell lysates to isolate nucleic acids, and for the speciation of proteins for mass spectrometry analysis. In this work, a novel and sensitive immobilization process was developed for examination of protein mixtures by combining MALDI-ToF-MS and nLC-TIMS-ToF-MS/MS systems. To achieve these goals, magnetic nanoparticles (MPs) were prepared via thermal coprecipitation reaction under alkaline condition. The MPs were grafted with a silica layer (i.e., 3-(2,3-epoxypropoxy) propyltrimethoxysilane; EPTES) containing reactive epoxy groups. Then, the silica-grafted magnetic particles were coated with a long chain hydrophilic poly(ethylene glycol) diamine polymer (PEGDAP). The prepared materials were characterized by the Brunauer-Emmett-Teller (BET) method, X-ray diffraction (XRD), scanning electron microscopy (SEM) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The VSM data show that the MPs@EPTES@PEGDAP has paramagnetic performance with a saturation magnetization of approximately 32.3 emu g-1. Proteinase K (EC 3.4.21.64) was covalently immobilized on the MPs@EPTES by reaction of its epoxy groups with amine groups of the enzyme. On the other hand, the ProK was immobilized on the MPs@EPTES@PEGDAP after activation with glutaraldehyde and the immobilization reaction was realized by the coupling reaction between aldehyde groups of the support and amine groups of the enzyme. The amounts of immobilized ProK on the MPs@EPTES and MPs@EPTES@PEGDAP were found to be 27.4 and 19.6 mg g-1and the retained activities were determined to be 29 and 87%, respectively. For the first time, some important features such as thermal and storage stabilities, reusability and potential use in protein speciation for mass spectrometry-based techniques were also evaluated. For examples, after six weeks of storage at 4 °C, the immobilized ProK on the MPs@EPTES@PEGDAP-ProK still maintained 59% of its initial activity. However, at the end of the six-week storage period, its free counterpart had lost all of its initial activity. The immobilized ProK was also utilized for degradation and identification of model proteins (i.e., α-2-HS glycoprotein, ß-casein, bovine serum albumin and immunoglobulin). After enzymatic treatment, the digested peptides were analyzed and mapped by using nLC-TIMS-ToF-MS/MS systems.

N-glycan profiling of papillary thyroid carcinoma tissues by MALDI-TOF-MS.

Papillary thyroid carcinoma (PTC) is a type of thyroid cancer whose incidence rate has increased recently all over the world. Glycosylation is a crucial post-translational modification (PTM) for the regulation of thyroid hormone synthesis in thyroid glands. However, our knowledge regarding the N-glycosylation change in PTC is limited. To the best of our knowledge, this is the first study to profile glycans in PTC tissues by mass spectrometry. Herein, we have analyzed the N-glycans of formalin-fixed paraffin-embedded (FFPE) tissues of patients diagnosed with PTC in a matched case-control study. Using MALDI-TOF(/TOF)-MS, 35 enzymatically released N-glycan compositions were characterized. The statistical analyses showed significant differences including six N-glycan compositions (p < 0.001) between patients and controls. It was determined that four of them (H5N4E1, H5N4F1E1, H5N4F1L1E1 and H5N4F1E2, E: α2,6-linked sialic acid; L: α2,3-linked sialic acid) were up-regulated in PTC tissues, whereas two N-glycans (H8N2 and H9N2) found to be down-regulated. Besides, a significant difference was found in six different N-glycan traits. Variants of PTC (follicular, classical, hurtle cell) were also studied to define specific N-glycan change for each variant.

Poly(amidoamine) dendrimer-coated magnetic nanoparticles for the fast purification and selective enrichment of glycopeptides and glycans.

Glycosylated proteins modulate various important functions of organisms. To reveal the functions of glycoproteins, in-depth characterization studies are necessary. Although mass spectrometry is a very efficient tool for glycoproteomic and glycomic studies, efficient sample preparation methods are required prior to analyses. In the study, poly(amidoamine) dendrimer-coated magnetic nanoparticles were presented for the specific enrichment and fast purification of glycopeptides and glycans. The enrichment and purification performance of the developed method was evaluated both at the glycopeptide, and the glycan level using several standard glycoprotein digests and released glycan samples. The poly(amidoamine) dendrimer-coated magnetic nanoparticles not only showed selective affinity (Immunoglobulin G/Bovine Serum Albumin, 1/10 by weight) to glycopeptides and released glycans but also good sensitivity (0.4 ng/µL for Immunoglobulin G) for glycoproteomic and glycomic applications. Thirty-five glycopeptides of Immunoglobulin G were detected after enrichment with poly(amidoamine) dendrimer-coated magnetic nanoparticles. In addition, 55 18 O tagged deamidated glycopeptides belonging to human plasma glycoproteome were confirmed. Finally, fifty 2-aminobenzoic acid, and 30 procainamide-labelled human plasma N-glycans released from human plasma glycoproteins were determined after purifications. The results indicate that the proposed enrichment and purification method using poly(amidoamine) dendrimer-coated magnetic nanoparticles could be simply adjusted to sample preparation methods.