n-glycan-profiling-glycoproteins-hydrophilic-interaction-liquid

<em>N-</em>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 ...

Karabuk University

Enrichment and/or purification of intact glycopeptides and derivatized glycans are essential for mass spectrometry-based glycoproteomics and glycomics because of analytical challenges observed in the analysis. Here, a titania-based material was synthesized using a facile sol-gel method for the efficient analysis of glycopeptides and glycans. The glycopeptide enrichment efficacy of the titania-based sol-gel material was evaluated by the enrichment of IgG N-glycopeptides from their proteolytic products. The selectivity and sensitivity (0.5 fmol μL) of the titania-based sol-gel material was found to be quite high when compared with commercial TiO. A pipette-tip application for rapid glycoproteomic and glycomic analysis was developed by packing titania-based material into the pipette tips. The released and derivatized N-glycans of human plasma, IgG and human transferrin were successfully purified by titania glyco-purification tips for the analysis by MALDI-MS. The enrichment performance of the titania glyco-purification tip was tested using human plasma digest as a real-world complex sample and, 112 N-glycopeptides from human plasma glycoproteins were detected by the analysis of enriched samples by nLC-QTOF-MS/MS. Finally, the structural characterization of the material was achieved in detail using various characterization approaches.

A New titania glyco-purification tip for the fast enrichment and efficient analysis of glycopeptides and glycans by MALDI-TOF-MS.

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  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.

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

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.

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

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. 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 gand 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, β-casein, bovine serum albumin and immunoglobulin). After enzymatic treatment, the digested peptides were analyzed and mapped by using nLC-TIMS-ToF-MS/MS systems.

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

Human milk is the optimal source of infant nutrition. Among many other health benefits, human milk can stimulate the development of a -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 . However, the bifidogenicity of human milk remains unparalleled. Dietary 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-β--acetylglucosaminidase enzyme (EndoBI-1) from  subspecies , in releasing 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.

Haci Mehmet Kayili

Department of Biomedical Engineering

Karabuk University

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

Haci Mehmet Kayili

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Engineering

Karabuk University

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

Department of Biomedical Engineering