The study of protein O-glycosylation is important in biological research as O-glycans have been reported to regulate a multitude of molecular and cell biology processes occurring in cancer. It is... Show moreThe study of protein O-glycosylation is important in biological research as O-glycans have been reported to regulate a multitude of molecular and cell biology processes occurring in cancer. It is known that alterations in O-glycosylation are involved in the development and progression of cancer. Their easy accessibility makes in vitro established cell lines suitable and useful models for studying biological mechanisms in disease. However, the O-glycosylation analysis of large numbers of samples, as required in systems biology and biomarker discovery studies, is often challenging. In the present study, O-glycans from three human colorectal cancer cell lines and two human pancreatic cancer cell lines were released by semi-automated, high throughput reductive beta-elimination and analysed using ultrahigh resolution MALDI-FT-ICR MS. Automated data integration and processing was performed using MassyTools, where the analyte was automatically included for relative quantitation based on a range of selection criteria including signal-to-noise ratio, mass error and isotopic pattern quality scores. A total of 126 O-glycan compositions, ranging from a single monosaccharide to large oligosaccharides exhibiting complex glycan motifs, were detected. The use of ultrahigh resolution MALDI-FTICR MS enabled glycan identification and quantitation in the matrix region of the spectrum. This approach has the potential to be used for O-glycosylation analysis of large numbers of samples, such as patient sample cohorts. Show less
A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories.Glycosylation is a topic of intense current... Show moreA broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories.Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods. Show less
The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes.... Show moreThe study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as bio-pharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated. Show less
Kotsias, M.; Blanas, A.; Vliet, S.J. van; Pirro, M.; Spencer, D.I.R.; Kozak, R.P. 2019
The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes.... Show moreThe study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated. Show less
Blanas, A.; Cornelissen, L.A.M.; Kotsias, M.; Horst, J.C. van der; Vrugt, H.J. van de; Kalay, H.; ... ; Vliet, S.J. van 2018
Aberrant fucosylation in cancer cells is considered as a signature of malignant cell transformation and it is associated with tumor progression, metastasis and resistance to chemotherapy.... Show moreAberrant fucosylation in cancer cells is considered as a signature of malignant cell transformation and it is associated with tumor progression, metastasis and resistance to chemotherapy. Specifically, in colorectal cancer cells, increased levels of the fucosylated Lewisx antigen are attributed to the deregulated expression of pertinent fucosyltransferases, like fucosyltransferase 4 (FUT4) and fucosyltransferase 9 (FUT9). However, the lack of experimental models closely mimicking cancer-specific regulation of fucosyltransferase gene expression has, so far, limited our knowledge regarding the substrate specificity of these enzymes and the impact of Lewisx synthesis on the glycome of colorectal cancer cells. Therefore, we sought to transcriptionally activate the Fut4 and Fut9 genes in the well-known murine colorectal cancer cell line, MC38, which lacks expression of the FUT4 and FUT9 enzymes. For this purpose, we utilized a physiologically relevant, guide RNA-based model of de novo gene expression, namely the CRISPR-dCas9-VPR system. Induction of the Fut4 and Fut9 genes in MC38 cells using CRISPR-dCas9-VPR resulted in specific neo-expression of functional Lewisx antigen on the cell surface. Interestingly, Lewisx was mainly carried by N-linked glycans in both MC38-FUT4 and MC38-FUT9 cells, despite pronounced differences in the biosynthetic properties and the expression stability of the induced enzymes. Moreover, Lewisx expression was found to influence core-fucosylation, sialylation, antennarity and the subtypes of N-glycans in the MC38-glycovariants. In conclusion, exploiting the CRISPR-dCas9-VPR system to augment glycosyltransferase expression is a promising method of transcriptional gene activation with broad application possibilities in glycobiology and oncology research. Show less
Changes in both N- and O-glycosylation have been associated with many states of health and disease, providing prognostic and diagnostic information. O-glycans have been relatively little studied... Show moreChanges in both N- and O-glycosylation have been associated with many states of health and disease, providing prognostic and diagnostic information. O-glycans have been relatively little studied through lack of suitable analytical tools but the evidence is that they could be promising biomarkers. The release and analysis of O-glycans remains very challenging. The main reason for this is that there is no enzyme available for universal O-glycan release. Therefore, chemical release is the most effective method. Unfortunately all of the O-glycan release methods show a side reaction known as “peeling”. Peeling is well known problem when performing the O-glycan release and often results in poor repeatability. The aim of the work described in this thesis was to develop or improve methods for the release of O-glycans with free reducing termini with high yield and limited degradation. The development of sample preparation techniques for O-glycan release (in their nonreduced form) by hydrazinolysis are described. The use of procainamide for fluorescent labelling of glycans followed by HILIC-UHPLC-ESI-MS/MS analysis is introduced. The application of the improved protocols for O-glycan release by hydrazinolysis combined with developed procainamide labelling system followed by HILIC-UHPLC-ESI-MS/MS analysis for evaluation of O-glycosylation changes in human saliva. Show less
The study of saliva O-glycosylation is receiving increasing attention due to the potential of glycans for disease biomarkers, but also due to easy access and non-invasive collection of saliva as... Show moreThe study of saliva O-glycosylation is receiving increasing attention due to the potential of glycans for disease biomarkers, but also due to easy access and non-invasive collection of saliva as biological fluid. Saliva is rich in glycoproteins which are secreted from the bloodstream or produced by salivary glands. Mucins, which are highly O-glycosylated proteins, are particularlyabundant in human saliva. Their glycosylation is associated with blood group and secretor status, and represents a reservoir of potential disease biomarkers. This study aims to analyse and compare O-glycans released from whole human mouth saliva collected 3 times a day from a healthy individual over a 5 days period. O-linked glycans were released by hydrazinolysis, labelled with procainamide and analysed by ultra-high performance liquid chromatography with fluorescence detection (UHPLC-FLR) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). The sample preparation method showed excellent reproducibility and can therefore be used for biomarker discovery. Our data demonstrates that the O-glycosylation in human saliva changes significantly during the day. These changes may be related to changes in the salivary concentrations of specific proteins. Show less
Monitoring glycoprotein therapeutics for changes in glycosylation throughout the drug's life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life,... Show moreMonitoring glycoprotein therapeutics for changes in glycosylation throughout the drug's life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life, safety, and immunogenicity. Biopharma companies are increasingly adopting Quality by Design (QbD) frameworks for measuring, optimizing, and controlling drug glycosylation. Permethylation of glycans prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a valuable tool for glycan characterization and for screening of large numbers of samples in QbD drug realization. However, the existing protocols for manual permethylation and liquid-liquid extraction (LLE) steps are labor intensive and are thus not practical for high-throughput (FIT) studies. Here we present a glycan permethylation protocol, based on 96-well microplates, that has been developed into a kit suitable for FIT work. The workflow is largely automated using a liquid handling robot and includes N-glycan release, enrichment of N-glycans, permethylation, and LLE. The kit has been validated according to industry analytical performance guidelines and applied to characterize biopharmaceutical samples, including IgG4 monoclonal antibodies (mAbs) and recombinant human erythropoietin (rhEPO). The HT permethylation enabled glycan characterization and relative quantitation with minimal side reactions: the MALDI-TOF-MS profiles obtained were in good agreement with hydrophilic liquid interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data. Automated permethylation and extraction of 96 glycan samples was achieved in less than 5 h and automated data acquisition on MALDI-TOF-MS took on average less than 1 min per sample. This automated and HT glycan preparation and permethylation showed to be convenient, fast, and reliable and can be applied for drug glycan profiling and clinical glycan biomarker studies. Show less
Immunoglobulin A (IgA) is a glycoprotein of which altered glycosylation has been associated with several pathologies. Conventional methods for IgA N- and O-glycosylation analysis are tedious, thus... Show moreImmunoglobulin A (IgA) is a glycoprotein of which altered glycosylation has been associated with several pathologies. Conventional methods for IgA N- and O-glycosylation analysis are tedious, thus limiting such analyses to small sample sizes. Here we present a high-throughput strategy for the simultaneous analysis of serum-derived IgA1 N- and O-glycopeptides using matrix-assisted laser/ desorption ionisation Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry (MS). Six non-fucosylated diantennary complex type glycoforms were detected on the Asn144-containing glycopeptide. Thirteen distinct glycoforms were identified for the Asn340-containing tailpiece glycopeptide, mainly of the diantennary complex type, and low amounts of triantennary glycoforms. Simultaneously with these N-glycopeptides, 53 compositional glycoforms of the hinge region O-glycopeptide were profiled in a single high resolution MALDI-FTICR spectrum. Since many pregnancy associated changes have been recognized for immunoglobulin G, we sought to demonstrate the clinical applicability of this method in a cohort of 29 pregnant women, from whom samples were collected at three time points during pregnancy and three time points after delivery. Pregnancy associated changes of N-glycan bisection were different for IgA1 as compared to IgG-Fc described earlier. We foresee further applications of the developed method for larger patient cohorts to study IgA N- and O-glycosylation changes in pathologies. Show less
