Automated high throughput IgG N-glycosylation sample preparation method development on the Tecan Freedom EVO platform.

Introduction: Glycomics, focusing on the role of glycans in biological processes, particularly their influence on the folding, stability and receptor interactions of glycoconjugates like antibodies, is vital for our understanding of biology. Changes in immunoglobulin G (IgG) N-glycosylation have been associated with various physiological and pathophysiological conditions. Nevertheless, time-consuming manual sample preparation is one of the limitations in the glycomics diagnostic implementation.

N-Glycosylation Profiles of Immunoglobulin G and Future Cardiovascular Events.

Background: Posttranslational glycosylation of IgG can modulate its inflammatory capacity through structural variations. We examined the association of baseline IgG N-glycans and an IgG glycan score with incident cardiovascular disease (CVD).

Methods: IgG N-glycans were measured in 2 nested CVD case-control studies: JUPITER (Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin; NCT00239681; primary prevention; discovery; Npairs=162); and TNT trial (Treating to New Targets; NCT00327691; secondary prevention; validation; Npairs=397).

Mapping of the gene network that regulates glycan clock of ageing.

Glycans are an essential structural component of immunoglobulin G (IgG) that modulate its structure and function. However, regulatory mechanisms behind this complex posttranslational modification are not well known. Previous genome-wide association studies (GWAS) identified 29 genomic regions involved in regulation of IgG glycosylation, but only a few were functionally validated.

Comparative analysis of transferrin and IgG N-glycosylation in two human populations.

Human plasma transferrin (Tf) N-glycosylation has been mostly studied as a marker for congenital disorders of glycosylation, alcohol abuse, and hepatocellular carcinoma. However, inter-individual variability of Tf N-glycosylation is not known, mainly due to technical limitations of Tf isolation in large-scale studies. Here, we present a highly specific robust high-throughput approach for Tf purification from human blood plasma and detailed characterization of Tf N-glycosylation on the level of released glycans by ultra-high-performance liquid chromatography based on hydrophilic interactions and fluorescence detection (HILIC-UHPLC-FLD), exoglycosidase sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

Total serum N-glycans associate with response to immune checkpoint inhibition therapy and survival in patients with advanced melanoma.

Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of melanoma and other cancers. However, no reliable biomarker of survival or response has entered the clinic to identify those patients with melanoma who are most likely to benefit from ICIs. Glycosylation affects proteins and lipids' structure and functions.

High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin.

Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR).

Immunoglobulin G N-Glycosylation Signatures in Incident Type 2 Diabetes and Cardiovascular Disease.

Objective: N-glycosylation is a functional posttranslational modification of immunoglobulins (Igs). We hypothesized that specific IgG N-glycans are associated with incident type 2 diabetes and cardiovascular disease (CVD).

Research Design And Methods: We performed case-cohort studies within the population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort (2,127 in the type 2 diabetes subcohort [741 incident cases]; 2,175 in the CVD subcohort [417 myocardial infarction and stroke cases]).

Seminal plasma N-glycome as a new biomarker of environmental exposure associated with semen quality.

Male infertility, a condition that has during the last decade raised significant concern, is a diagnostically demanding and socially sensitive topic. The number of unsolved issues on infertility etiology, especially potential environmental causes, in couples demonstrates the need for further investigations into infertility biomarkers. Semen parameters are often insufficient for reliable profiling of male infertility.

IgG N-glycome changes during the course of severe COVID-19: An observational study.

Background: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes a respiratory illness named coronavirus disease 2019 (COVID-19), which is one of the main global health problems since 2019. Glycans attached to the Fc portion of immunoglobulin G (IgG) are important modulators of IgG effector functions. Fc region binds to different receptors on the surface of various immune cells, dictating the type of immune response.

Developments and perspectives in high-throughput protein glycomics: enabling the analysis of thousands of samples.

Glycans expand the structural complexity of proteins by several orders of magnitude, resulting in a tremendous analytical challenge when including them in biomedical research. Recent glycobiological research is painting a picture in which glycans represent a crucial structural and functional component of the majority of proteins, with alternative glycosylation of proteins and lipids being an important regulatory mechanism in many biological and pathological processes. Since interindividual differences in glycosylation are extensive, large studies are needed to map the structures and to understand the role of glycosylation in human (patho)physiology.

Immunoglobulin G glycome composition in transition from premenopause to postmenopause.

Gonadal hormones affect immunoglobulin G (IgG) glycosylation, and the more proinflammatory IgG glycome composition might be one of the molecular mechanisms behind the increased proinflammatory phenotype in perimenopause. Using ultra-high-performance liquid chromatography, we analyzed IgG glycome composition in 5,080 samples from 1940 pre-, peri-, and postmenopausal women. Statistically significant decrease in galactosylation and sialylation was observed in postmenopausal women.

The Importance of Glycosylation in COVID-19 Infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently one of the major health problems worldwide. SARS-CoV-2 survival and virulence are shown to be impacted by glycans, covalently attached to proteins in a process of glycosylation, making glycans an area of interest in SARS-CoV-2 biology and COVID-19 infection. The SARS-CoV-2 uses its highly glycosylated spike (S) glycoproteins to bind to the cell surface receptor angiotensin-converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry.

Glycosylation of IgG Associates with Hypertension and Type 2 Diabetes Mellitus Comorbidity in the Chinese Muslim Ethnic Minorities and the Han Chinese.

Objectives: Hypertension and type 2 diabetes mellitus comorbidity (HDC) is common, which confers a higher risk of cardiovascular disease than the presence of either condition alone. Describing the underlying glycomic changes of immunoglobulin G (IgG) that predispose individuals to HDC may help develop novel protective immune-targeted and anti-inflammatory therapies. Therefore, we investigated glycosylation changes of IgG associated with HDC.

N-glycosylation profiling of Type 2 diabetes mellitus from baseline to follow-up: an observational study in a Ghanaian population.

The study sought to determine the patterns of N-glycan profiles among Type 2 diabetes mellitus (T2DM) patients over a 6-month period. Biochemical and clinical data were obtained from 253 T2DM patients at baseline and follow-up. Ultra-performance liquid chromatography and statistical methods were applied for N-glycan profiling.