The Impact of Protein Glycosylation on the Identification of Patients with Pediatric Appendicitis.

The identification of pediatric appendicitis is challenging due to the lack of specific markers thereby several factors are included in the diagnostic process such as abdominal pain, ultrasonography and altered laboratory parameters (C reactive protein, absolute neutrophil cell number and white blood cell number). The glycosylation pattern of serum N-glycome was analyzed in this study of 38 controls and 40 patients with pediatric appendicitis. The glycans were released by enzymatic deglycosylation followed by fluorescent labeling and solid-phase extraction.

Simplified Synthesis of the Amine-Functionalized Magnesium Ferrite Magnetic Nanoparticles and Their Application in DNA Purification Method.

For pathogens identification, the PCR test is a widely used method, which requires the isolation of nucleic acids from different samples. This extraction can be based on the principle of magnetic separation. In our work, amine-functionalized magnesium ferrite nanoparticles were synthesized for this application by the coprecipitation of ethanolamine in ethylene glycol from Mg(II) and Fe(II) precursors.

Preparation and Optical Study of 1-Formamido-5-Isocyanonaphthalene, the Hydrolysis Product of the Potent Antifungal 1,5-Diisocyanonaphthalene.

Aromatic isocyanides have gained a lot of attention lately as promising antifungal and anticancer drugs, as well as high-performance fluorescent analytical probes for the detection of toxic metals, such as mercury, even in vivo. Since this topic is relatively new and aromatic isocyanides possess unique photophysical properties, the understanding of structure-behavior relationships and the preparation of novel potentially biologically active derivatives are of paramount importance. Here, we report the photophysical characterization of 1,5-diisocyanonaphthalene (DIN) backed by quantum chemical calculations.

The Alterations of Serum N-glycome in Response to SARS-CoV-2 Vaccination.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a global concern since its outbreak in 2019, with one of the main solutions being vaccination. Altered glycosylation has been described in patients after SARS-CoV-2 infection, while the effect of vaccination on serum glycoproteins remained unexplored. In this study, total serum glycosylation was analyzed in patients after SARS-CoV-2 infection and/or mRNA vaccination in order to identify potential glycosylation-based alterations.

A Simplified and Efficient Method for Production of Manganese Ferrite Magnetic Nanoparticles and Their Application in DNA Isolation.

A simplified, fast, and effective production method has been developed for the synthesis of manganese ferrite (MnFeO) magnetic nanoparticles (MNPs). In addition to the wide applicability of MnFeO MNPs, this work also reports their application in DNA isolation for the first time. An ultrasonic-cavitation-assisted combustion method was applied in the synthesis of MnFeO MNPs at different furnace temperatures (573 K, 623 K, 673 K, and 773 K) to optimize the particles' properties.

NH-Functionalized Magnetic Nanoparticles for the -Glycomic Analysis of Patients with Multiple Sclerosis.

Glycosylation is vital for well-functioning glycoproteins and is reportedly altered in chronic inflammatory disorders, including multiple sclerosis (MS). High-throughput quantitative measurement of protein glycosylation is challenging, as glycans lack fluorophore groups and require fluorescent labeling. The attachment of fluorescent tags to each glycan moiety necessitates sample clean-up for reliable quantitation.

Sonochemical Combined Synthesis of Nickel Ferrite and Cobalt Ferrite Magnetic Nanoparticles and Their Application in Glycan Analysis.

The combination of the sonochemical activation of Ni(NO) and Co(NO) in the presence of Fe(NO) and polyethylene glycol and consecutive heat treatment of the formed metal hydroxides offers a cheap and efficient method for the preparation of nickel ferrite and cobalt ferrite magnetic nanoparticles, which can be successfully applied in the selective capture of fluorescently derivatized N-glycans from human serum. XRD measurement revealed that, besides the ferrite phase, nickel and cobalt oxides also form during heat treatment. The amount of simple metal oxides can be well controlled by the temperature of the heat treatment, since increasing temperature yielded higher spinel content.

The Analysis of Human Serum N-Glycosylation in Patients with Primary and Metastatic Brain Tumors.

The identification of patients with different brain tumors is solely built on imaging diagnostics, indicating the need for novel methods to facilitate disease recognition. Glycosylation is a chemical modification of proteins, reportedly altered in several inflammatory and malignant diseases, providing a potential alternative route for disease detection. In this paper, we report the quantitative analysis of serum N-glycosylation of patients diagnosed with primary and metastatic brain tumors.

Clinical Features of Parkinson's Disease: The Evolution of Critical Symptoms.

Parkinson's disease (PD) is a multi-attribute neurodegenerative disorder combining motor and nonmotor symptoms without well-defined diagnostic clinical markers. The presence of primary motor features (bradykinesia, rest tremor, rigidity and loss of postural reflexes) are the most characteristic signs of PD that are also utilized to identify patients in current clinical practice. The successful implementation of levodopa treatment revealed that nonmotor features are the main contributors of patient disability in PD, and their occurrence might be earlier than motor symptoms during disease progression.

Analysis of cetuximab N-Glycosylation using multiple fractionation methods and capillary electrophoresis mass spectrometry.

Site-specific glycosylation of Cetuximab was characterized in this study using multiple fractionation methods and capillary electrophoresis coupled to mass spectrometry (CE-MS) based glycomics. IdeS digested Cetuximab with subsequent reduction was fractionated using reversed-phase chromatography resulting in 3 fragments; Fd, Lc and Fc/2. Glycan release of the different fragments was performed in O enriched water providing the possible quantification of site occupancy.

Purification of Fluorescently Derivatized N-Glycans by Magnetic Iron Nanoparticles.

A novel glycoanalytical approach was developed in this study for the purification of fluorescently derivatized N-glycans. Polyethylene glycol (PEG) modified iron-nanoparticles were synthetized by the combination of sonochemical treatment and combustion method. The prepared nanomaterials were applied for a systematic clean-up optimization to maximize purification efficiency of 2-AA labelled glycans.

Serum N-Glycosylation in Parkinson's Disease: A Novel Approach for Potential Alterations.

In this study, we present the application of a novel capillary electrophoresis (CE) method in combination with label-free quantitation and support vector machine-based feature selection (support vector machine-estimated recursive feature elimination or SVM-RFE) to identify potential glycan alterations in Parkinson's disease. Specific focus was placed on the use of neutral coated capillaries, by a dynamic capillary coating strategy, to ensure stable and repeatable separations without the need of non-mass spectrometry (MS) friendly additives within the separation electrolyte. The developed online dynamic coating strategy was applied to identify serum N-glycosylation by CE-MS/MS in combination with exoglycosidase sequencing.

Multi-site N-Glycan mapping study 2: UHPLC.

In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N-glycosylation analysis using capillary electrophoresis of APTS-labeled N-glycans were presented.

Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry.

Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein-derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statistical analysis when optical detection methods are used. The approach outlined herein describes the utilization of stable isotope variants of commonly used fluorescent tags that allow for mass-based glycan identification and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE).

Quantitative glycomics using liquid phase separations coupled to mass spectrometry.

Post-translational modification of proteins by the attachment of glycans is governed by a variety of highly specific enzymes and is associated with fundamental impacts on the parent protein's physical, chemical and biological properties. The inherent connection between cellular physiology and specific glycosylation patterns has been shown to offer potential for diagnostic and prognostic monitoring of altered glycosylation in the disease state. Conversely, glycoprotein based biopharmaceuticals have emerged as dominant therapeutic strategies in the treatment of intricate diseases.

Quantitative twoplex glycan analysis using C and C stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry.

Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS).

Multi-Site N-glycan mapping study 1: Capillary electrophoresis - laser induced fluorescence.

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.

Comparative glycoprofiling of HIV gp120 immunogens by capillary electrophoresis and MALDI mass spectrometry.

The human immunodeficiency virus (HIV) envelope glycoprotein (Env) is the primary antigenic feature on the surface of the virus and is of key importance in HIV vaccinology. Vaccine trials with the gp120 subunit of Env are ongoing, with the recent RV144 trial showing moderate efficacy. gp120 is densely covered with N-linked glycans that are thought to help evade the host's humoral immune response.

Sample preparation for N-glycosylation analysis of therapeutic monoclonal antibodies by electrophoresis.

There are a considerable number of biopharmaceuticals that have been approved for clinical use in the past decade. Over half of these new generation drugs are glycoproteins, such as monoclonal antibodies or other recombinant glycoproteins, which are mostly produced in mammalian cell lines. The linked carbohydrate moieties affect not only their physicochemical properties and thermal stability but also crucial features like receptor-binding activity, circulating half-life, as well as immunogenicity.

Combination of IgG N-glycomics and corresponding transcriptomics data to identify anti-TNF-α treatment responders in inflammatory diseases.

Prediction of responsiveness in biological therapies is an important and challenging issue in different diseases. Analyzing glycosylation pattern changes of key serum glycoproteins is one of the possible avenues to follow disease remission. The aim of this study was to investigate the changes of serum IgG glycoforms in Crohn's disease (CD) and rheumatoid arthritis patients in response to antitumor necrosis factor alpha (anti-TNF-α) treatment.

Rapid magnetic bead based sample preparation for automated and high throughput N-glycan analysis of therapeutic antibodies.

Full automation to enable high throughput N-glycosylation profiling and sequencing with good reproducibility is vital to fulfill the contemporary needs of the biopharmaceutical industry and requirements of national regulatory agencies. The most prevalently used glycoanalytical methods of capillary electrophoresis and hydrophilic interaction liquid chromatography, while very efficient, both necessitate extensive sample preparation and cleanup, including glycoprotein capture, N-glycan release, fluorescent derivatization, purification, and preconcentration steps during the process. Currently used protocols to fulfill these tasks require multiple centrifugation and vacuum-centrifugation steps, making liquid handling robot mediated automated sample preparation difficult and expensive.

Analysis of haptoglobin N-glycome alterations in inflammatory and malignant lung diseases by capillary electrophoresis.

A CE-based method was introduced to compare the N-glycosylation profile of haptoglobin in normal and pathologic conditions. To assess the biomarker potential of glycosylation changes in various lung diseases, haptoglobin was isolated from plasma samples of healthy, pneumonia, chronic obstructive pulmonary disease, and lung cancer patients by means of two haptoglobin-specific monoclonal antibodies. Haptoglobin N-glycans were then enzymatically released, fluorescently labeled, and profiled by CE.

Peripheral blood gene expression and IgG glycosylation profiles as markers of tocilizumab treatment in rheumatoid arthritis.

Objective: Tocilizumab, a humanized anti-interleukin-6 receptor monoclonal antibody, has recently been approved as a biological therapy for rheumatoid arthritis (RA) and other diseases. It is not known if there are characteristic changes in gene expression and immunoglobulin G glycosylation during therapy or in response to treatment.

Methods: Global gene expression profiles from peripheral blood mononuclear cells of 13 patients with RA and active disease at Week 0 (baseline) and Week 4 following treatment were obtained together with clinical measures, serum cytokine levels using ELISA, and the degree of galactosylation of the IgG N-glycan chains.