Serum Glycome as a Diagnostic and Prognostic Factor in Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a risk factor for both mother and fetus/neonate during and after the pregnancy. Inconsistent protocols and cumbersome screening procedures warrant the search for new and easily accessible biomarkers. We investigated a potential of serum N-glycome to differentiate between healthy pregnant women (n = 49) and women with GDM (n = 53) using a lectin-based microarray and studied the correlation between the obtained data and parameters of glucose and lipid metabolism. Four out of 15 lectins used were able to detect the differences between the control and GDM groups in fucosylation, terminal galactose/N-acetylglucosamine (Gal/GlcNAc), presence of Galα1,4Galß1,4Glc (Gb3 antigen), and terminal α2,3-sialylation with AUC values above 60%. An increase in the Gb3 antigen and α2,3-sialylation correlated positively with GDM, whereas the amount of fucosylated glycans correlated negatively with the content of terminal Gal/GlcNAc. The content of GlcNAc oligomers correlated with the highest number of blood analytes, indices, and demographic characteristics, but failed to discriminate between the groups. The presence of terminal Gal residues correlated positively with the glucose levels and negatively with the LDL levels in the non-GDM group only. The results suggest fucosylation, terminal galactosylation, and the presence of Gb3 antigen as prediction markers of GDM.

Current look at the most promising proteomic and glycomic biomarkers of bladder cancer.

BACKGROUND: Bladder cancer (BC) belongs to the most frequent cancer types. The diagnostic process is still long and costly, with a high percentage of false-positive or -negative results. Due to the cost and lack of effectiveness, older methods need to be supplemented or replaced by a newer more reliable method. In this regard, proteins and glycoproteins pose high potential. METHODS: We performed an online search in PubMed/Medline, Scopus, and Web of Science databases to find relevant studies published in English up until May 2023. If applicable, we set the AUC threshold to 0.90 and sensitivity/specificity (SN/SP) to 90%. FINDINGS: Protein and glycoprotein biomarkers are a demonstrably viable option in BC diagnostics. Cholinesterase shows promise in progression-free survival. BLCA-4, ORM-1 along with HTRA1 in the detection of BC. Matrix metallopeptidase 9 exhibits potential for stratification of muscle-invasive subtypes with high negative predictive value for aggressive phenotypes. Distinguishing non-muscle invasive subtypes benefits from Keratin 17. Neu5Gc-modified UMOD glycoproteins pose potential in BC diagnosis, while fibronectin, laminin-5, collagen type IV, and lamprey immunity protein in early detection of BC.

Identification and evaluation of antiviral activity of novel compounds targeting SARS-CoV-2 virus by enzymatic and antiviral assays, and computational analysis.

The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases Mpro and PLpro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC50 values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC50 against PLpro at approximately 10-fold higher micromolar concentrations. Although originally developed as PLpro inhibitors, the comparison between IC50 and EC50 of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.

O-glycoprofiling of Serum Apolipoprotein C-III in Colorectal Cancer.

BACKGROUND: Aberrant glycosylation is a hallmark of cancer and thereby has an excellent potential for the discovery of novel biomarkers. Impairments in the glycan composition of lipoproteins impact their functional properties and can be associated with various diseases, including cancer. This research is still in its infancy; however, it can lead to the development of new diagnostic and disease stratification approaches as well as therapeutic strategies. Therefore, we aimed to evaluate anomalies in O-glycosylation of apolipoprotein C-III (apoC-III) in colorectal carcinoma (CRC) patients' sera, in comparison with sera from healthy individuals, and assess the disparities of O-glycoforms on apoC-III in CRC. METHODS: The choice of patients (n = 42) was based on the same tumor type (adenocarcinoma) and tumor size (T3), without or with inconsiderable lymph node infiltration. Patients with comorbidities were excluded from the study. The control healthy individuals (n = 40) were age- and sex-matched with patients. We used an approach based on the MALDI-TOF MS in linear positive ion mode, allowing simple analysis of O-glycosylation on intact apoC-III molecules in the serum samples directly, without the need for specific protein isolation. This approach enables relatively simple and high-throughput analysis. RESULTS: In CRC patients' sera samples, we observed significantly elevated apoC-III sialylation. Fully sialylated (disialylated) O-glycans had 1.26 times higher relative abundance in CRC samples compared to controls with a p-value of Mann-Whitney U test of 0.0021. CONCLUSIONS: We found altered O-glycosylation of apoC-III in the serum of CRC patients. However, it can be non-specific as it may be associated with another process such as ongoing inflammation. Therefore, to establish it as a potential novel non-invasive biomarker for CRC in suspected patients, further studies interrogating the changes in apoC-III O-glycosylation and the robustness of this biomarker need to be performed and evaluated.

Universal protocol for the wafer-scale manufacturing of 2D carbon-based transducer layers for versatile biosensor applications.

In this manuscript, we present a comprehensive fabrication protocol for high-performance graphene oxide (GO) sensor concepts. It is suitable for a variety of biosensing applications and contains the essential process steps, starting with vapor phase evaporation for siloxane monolayers, followed by spin-coating of GO as a nanometer-thin transducer with exceptional homogeneity and micromechanical surface methods which enable seamless transformation of GO transducers to be desired micro and nano dimensions. In addition to linking basic research and innovative sensor concepts with an outlook for commercial applications of point-of-care systems for early-stage diagnostics, the authors consider it necessary to take a closer look at the manufacturing processes to create more transparency and clarity, to manufacture such specific sensor concepts systematically. The detailed manufacturing approaches are intended to motivate practitioner to explore and improve this GO-based key technology. This process development is illustrated below using the manufacturing methods for three types of sensors, namely sensors based on i) surface plasmon resonance spectroscopy (SPR), ii) impedance spectroscopy and iii) bio-field effect transistors (ISFETs). The obtained results in this work prove successful GO sensor productions by achieving:•Uniform and stable immobilization of GO thin films,•High yield of sensor units on a wafer scale, here up to 96 %,•Promising integration potential for various biomedical sensor concepts to early-stage diagnostic.

A Holistic 4D Approach to Optimize Intrinsic and Extrinsic Factors Contributing to Variability in Microarray Biosensing in Glycomics.

Protein-carbohydrate interactions happen to be a crucial facet of biology, discharging a myriad of functions. Microarrays have become a premier choice to discern the selectivity, sensitivity and breadth of these interactions in a high-throughput manner. The precise recognition of target glycan ligands among the plethora of others is central for any glycan-targeting probe being tested by microarray analyses. Ever since the introduction of the microarray as an elemental tool for high-throughput glycoprofiling, numerous distinct array platforms possessing different customizations and assemblies have been developed. Accompanying these customizations are various factors ushering variances across array platforms. In this primer, we investigate the influence of various extrinsic factors, namely printing parameters, incubation procedures, analyses and array storage conditions on the protein-carbohydrate interactions and evaluate these factors for the optimal performance of microarray glycomics analysis. We hereby propose a 4D approach (Design-Dispense-Detect-Deduce) to minimize the effect of these extrinsic factors on glycomics microarray analyses and thereby streamline cross-platform analyses and comparisons. This work will aid in optimizing microarray analyses for glycomics, minimize cross-platform disparities and bolster the further development of this technology.

Significance of 1,25-Dihydroxyvitamin D3 on Overall Mortality in Peritoneal Dialysis Patients with COVID-19.

In previous publications, we pointed out the importance of mannosylation of fibrinogen for the development of cardiovascular complications and fucosylation as a predictor of peritoneal membrane dysfunction in patients on peritoneal dialysis (PD). After a follow-up period of 30 months from the onset of the COVID-19 pandemic, we evaluated the significance of 1,25-dihydroxyvitamin D3 (calcitriol) therapy, primary disease, biochemical and hematologic analyzes, and previously performed glycan analysis by lectin-based microarray as predictors of mortality in this patient group. After univariate Cox regression analysis, diabetes mellitus (DM) and calcitriol therapy were found to be potential predictors of mortality. Additional multivariate Cox regression analysis confirmed that only DM was a predictor of mortality. Nevertheless, the use of calcitriol in therapy significantly reduced mortality in this patient group, as shown by the Kaplan-Meier survival curve. The presence of DM as a concomitant disease proved to be a strong predictor of fatal outcome in PD patients infected with SARS-CoV-2. This is the first study to indicate the importance and beneficial effect of calcitriol therapy on survival in PD patients with COVID-19 infection. In addition, this study points to the possibility that adverse thrombogenic events observed in PD patients during the pandemic may be caused by aberrant fibrinogen glycosylation.

Alterations in the Glycan Composition of Serum Glycoproteins in Attention-Deficit Hyperactivity Disorder.

Changes in protein glycosylation are associated with most biological processes, and the importance of glycomic analysis in the research of disorders is constantly increasing, including in the neurodevelopmental field. We glycoprofiled sera in 10 children with attention-deficit hyperactivity disorder (ADHD) and 10 matching healthy controls for 3 types of samples: whole serum, sera after depletion of abundant proteins (albumin and IgG), and isolated IgG. The analytical methods used were a lectin-based glycoprotein microarray enabling high-throughput glycan analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) as a standard method for the identification of glycan structures. For microarray analysis, the samples printed on microarray slides were incubated with biotinylated lectins and detected using the fluorescent conjugate of streptavidin by a microarray scanner. In the ADHD patient samples, we found increased antennary fucosylation, decreased di-/triantennary N-glycans with bisecting N-acetylglucosamine (GlcNAc), and decreased α2-3 sialylation. The results obtained by both independent methods were consistent. The study's sample size and design do not allow far-reaching conclusions to be drawn. In any case, there is a strong demand for a better and more comprehensive diagnosis of ADHD, and the obtained results emphasize that the presented approach brings new horizons to studying functional associations of glycan alterations in ADHD.

The Application of HPLC-FLD and NMR in the Monitoring of Therapy Efficacy in Alpha-Mannosidosis.

BACKGROUND: Alpha-mannosidosis is a rare lysosomal storage disorder, caused by decreased activity of α-D-mannosidase. This enzyme is involved in the hydrolysis of mannosidic linkages in N-linked oligosaccharides. Due to the mannosidase defect, undigested mannose-rich oligosaccharides (Man2GlcNAc - Man9GlcNAc) accumulating in cells are excreted in large quantities in urine. METHODS: In this work, we determined the levels of urinary mannose-rich oligosaccharides in a patient subjected to novel enzyme replacement therapy. Urinary oligosaccharides were extracted using solid phase extraction (SPE), labeled by fluorescent tag 2-aminobenzamide, and quantified by high-performance liquid chromatography (HPLC) with fluorescence detector (FLD). The identity of peaks was determined by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. In addition, the levels of urinary mannose-rich oligosaccharides were also quantified by 1H nuclear magnetic resonance (NMR) spectroscopy. The data were analyzed using one-tailed paired t-test and Pearson's correlation tests. RESULTS: Compared to levels before the administration of therapy, an approximately two-folds decrease in total mannose-rich oligosaccharides after one month of treatment was observed by NMR and HPLC. After four months, an approximately ten-folds significant decrease in total urinary mannose-rich oligosaccharides was detected, suggesting therapy effectiveness. A significant decrease in the levels of oligosaccharides with 7-9 mannose units was detected by HPLC. CONCLUSIONS: The application of both HPLC-FLD and NMR in quantification of oligosaccharide biomarkers is a suitable approach for monitoring of therapy efficacy in alpha-mannosidosis patients.

Prediction of Mortality in Patients on Peritoneal Dialysis Based on the Fibrinogen Mannosylation.

As we already reported, fibrinogen fucosylation emerged as a prognostic marker of peritoneal membrane function in end-stage renal disease (ESRD) patients on peritoneal dialysis. After a follow-up period of 18 months, we estimated the ability of employed lectins, as well as other biochemical parameters, to serve as mortality predictors in these patients. Following a univariate Cox regression analysis, ferritin, urea clearance, residual diuresis, hyperglycemia, and an increase in the signal intensity obtained with Galanthus nivalis lectin (GNL) emerged as potential mortality predictors, but additional multivariate Cox regression analysis pointed only to glucose concentration and GNL as mortality predictors. Higher signal intensity obtained with GNL in patients that died suggested the importance of paucimannosidic/highly mannosidic N-glycan structures on fibrinogen as factors that are related to unwanted cardiovascular events and all-cause mortality and can possibly be seen as a prediction tool. Altered glycan structures composed of mannose residues are expected to affect the reactivity of mannosylated glycoproteins with mannose-binding lectin and possibly the entire cascade of events linked to this lectin. Since patients with ESRD are prone to cardiovascular complications and the formation of atherosclerotic plaques, one can hypothesize that fibrinogen with increasingly exposed mannose residues may contribute to the unwanted events.

Investigation of the Potential of Selected Food-Derived Antioxidants to Bind and Stabilise the Bioactive Blue Protein C-Phycocyanin from Cyanobacteria Spirulina.

Blue C-phycocyanin (C-PC), the major Spirulina protein with innumerable health-promoting benefits, is an attractive colourant and food supplement. A crucial obstacle to its more extensive use is its relatively low stability. This study aimed to screen various food-derived ligands for their ability to bind and stabilise C-PC, utilising spectroscopic techniques and molecular docking. Among twelve examined ligands, the protein fluorescence quenching revealed that only quercetin, coenzyme Q10 and resveratrol had a moderate affinity to C-PC (Ka of 2.2 to 3.7 × 105 M-1). Docking revealed these three ligands bind more strongly to the C-PC hexamer than the trimer, with the binding sites located at the interface of two (αß)3 trimers. UV/VIS absorption spectroscopy demonstrated the changes in the C-PC absorption spectra in a complex with quercetin and resveratrol compared to the spectra of free protein and ligands. Selected ligands did not affect the secondary structure content, but they induced changes in the tertiary protein structure in the CD study. A fluorescence-based thermal stability assay demonstrated quercetin and coenzyme Q10 increased the C-PC melting point by nearly 5 °C. Our study identified food-derived ligands that interact with C-PC and improve its thermal stability, indicating their potential as stabilising agents for C-PC in the food industry.

Electrochemical Aptasensors for Parkinson's Disease Biomarkers Detection.

BACKGROUND: Biomarkers are characteristic molecules that can serve as indicators of biological process status or condition; here, they are being studied with special relevance to Parkinson's Disease (PD). This disease is a chronic neurodegenerative disorder very difficult to study given the site of pathology and due to a clinical phenotype that fluctuates over time. Currently, there is no definitive diagnostic test for Parkinson's Disease; thus, clinicians hope that the detection of crucial biomarkers will help in the symptomatic and presymptomatic diagnostics and provide surrogate endpoints to demonstrate the clinical efficacy of new treatments. METHODS: Electrochemical aptasensors are excellent analytical tools that are used in the detection of PD biomarkers, as they are portable, easy to use, and perform real-time analysis. RESULTS: In this review, we discuss the most important clinical biomarkers for PD, highlighting their physiological role and function in the disease. Herein, we review, for the first time, innovative aptasensors for the detection of current potential PD biomarkers based on electrochemical techniques and discuss future alternatives, including ideal analytical platforms for point-of-care diagnostics. CONCLUSION: These new tools will be critical not only in the discovery of sensitive, specific, and reliable biomarkers of preclinical PD, but also in the development of tests that can assist in the early detection and differential diagnosis of parkinsonian disorders and in monitoring disease progression. Various methods for fixing aptamers onto the sensor surfaces, enabling quantitative and specific PD biomarker detection present in synthetic and clinical samples, will also be discussed.

Features, modulation and analysis of glycosylation patterns of therapeutic recombinant immunoglobulin A.

Increasing the production of recombinant antibodies while ensuring high and stable protein quality remains a challenge in mammalian cell culture. This review is devoted to advances in the field of obtaining stable and optimal glycosylation of therapeutic antibodies based on IgA, as well as the subsequent issues of glycosylation control of glycoproteins during their production. Current studies also demonstrate a general need for a more fundamental understanding of the use of CHO cell-based producer cell lines, through which the glycoprofile of therapeutic IgA antibodies is produced and the dependence of glycosylation on culture conditions could be controlled. Optimization of glycosylation improves the therapeutic efficacy and can expand the possibilities for the creation of highly effective glycoprotein therapeutic drugs. Current status and trends in glycan analysis of therapeutic IgA, dominantly based on mass spectrometry and lectin microarrays are herein summarised as well.

Lectin-Based Protein Microarray for the Glycan Analysis of Colorectal Cancer Biomarkers: The Insulin-Like Growth Factor System.

Lectin-based protein microarrays are used for glycoprofiling of various kinds of biological samples. Here we describe lectin-based microarray assay in the reverse-phase format where glycoprotein samples are spotted onto microarray slide and then are incubated with set of lectins. This configuration allows high-throughput screening of a large cohort of samples by a set of lectins without need of separation of glycans from glycoproteins. We applied the described method for glycan analysis of glycoprotein biomarkers of colorectal cancer associated with the insulin-like growth factor system.

Lectin-based assay for the determination of the inhibition activity of small molecule inhibitors of neuraminidases.

Small molecules inhibitors of neuraminidases (NAs) are ones of the most prospective molecules proposed for the treatment of influenza viruses. The determination of their inhibition activity in vitro is an important step during the development of antiviral drugs. However, the analytical methods typically used for the evaluation of NA activity and inhibition (fluorescence-based assays using MUNANA substrate or thiobarbituric acid assay, TBA) may suffer from interferences caused by tested inhibitors as signal quenching or self-fluorescence, moreover in TBA are used toxic and carcinogenic reagents. The determination of the NA activity can be effectively performed by alternative methods based on lectin - glycan recognition, usually as enzyme-linked lectin assay (ELLA). We have adapted the ELLA assay to a lectin-based assay in a microplate format with fluorescence detection for determination of NA inhibitory activity. We optimized our protocol and the developed method was tested using four different small molecule NA inhibitors or potential NA inhibitors (DANA, zanamivir, quercetin and α-mangostin) with three bacterial NAs (from Clostridium perfringens, Vibrio cholerae and Arthrobacter ureafaciens), and the IC50 values for NA inhibitors were determined. The inhibition effect of DANA was observed for all 3 tested NAs (IC50 = 10.1 µM for V. cholerae, 13.4 µM for C. perfringens and 402.9 µM for A. ureafaciens, respectively) and of Zanamivir only for NA from V. cholerae (IC50 = 101.9 µM). For both quercetin and α-mangostin, no inhibition effect to the tested NAs was observed. The main advantages of herein described method are good sensitivity due to fluorescent signal detection, the absence of the interference caused by fluorescent signal quenching by tested inhibitors, the use of natural substrates (glycoproteins) and the avoiding the use of toxic reagents.

Fibrinogen Fucosylation as a Prognostic Marker of End-Stage Renal Disease in Patients on Peritoneal Dialysis.

Glycosylation may strongly affect protein structure and functions. A high risk of cardiovascular complications seen in patients with end-stage renal disease (ESRD) is, at least partly associated with delayed clot formation, increased clot strength, and delayed cloth lysis. Taking into consideration that fibrinogen mediates these processes, we isolated fibrinogen from the plasma from patients with ESRD on peritoneal dialysis (ESRD-PD), and examined glycosylation of native fibrinogen and its subunits by lectin-based microarray and lectin blotting. Compared to healthy controls, fibrinogen from patients had increased levels of A2BG2 and decreased levels of FA2 glycan. The distribution of glycans on individual chains was also affected, with the γ chain, responsible for physiological functions of fibrinogen (such as coagulation and platelet aggregation), being most prone to these alterations. Increased levels of multi-antennary N-glycans in ESRD-PD patients were also associated with the type of dialysis solutions, whereas an increase in the fucosylation levels was strongly related to the peritoneal membrane damage. Consequently, investigation of fibrinogen glycans can offer better insight into fibrinogen-related complications observed in ESRD-PD patients and, additionally, contribute to prognosis, choice of personalised therapy, determination of peritoneal membrane damage, and the length of utilization of peritoneum for dialysis.

Analytical techniques for multiplex analysis of protein biomarkers.

INTRODUCTION: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. AREAS COVERED: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. EXPERT COMMENTARY: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.

Influence of media composition on recombinant monoclonal IgA1 glycosylation analysed by lectin-based protein microarray and MALDI-MS.

Glycosylation of therapeutic glycoproteins significantly affects their physico-chemical properties, bioactivity and immunogenicity. The determination of glycan composition is highly important regarding their development and production. Therefore, there is a demand for analytical techniques enabling rapid and reliable glycoprofiling of therapeutic proteins. For the investigation of changes in glycan structures, we have employed two platforms: lectin-based protein microarray, and MALDI-MS. In lectin-based microarray analysis, the samples of IgA were printed on the microarray slide, incubated with the set of lectins with various specificity and evaluation of changes in glycosylation was based on differences in reactivity of samples with lectins. MALDI-MS was used for N-glycan analysis of IgA1 samples. IgAs are effective as therapeutic agents in defense against viruses that use sialic acid as a receptor. Dimeric IgA1 antibodies were produced by stable cell line IgA1/2G9 on the basal medium at different conditions (different supplementation and feeding) and we also evaluated the effect of different conditions on lactate production, which correlates with IgA productivity. Decrease of lactate levels was observed during supplementation with succinic acid, asparagine, or with mannose feeding. We found by lectin-based microarray analysis that the metabolic shift from glutamine to asparagine or feeding with glucose caused increase of high mannose type glycans what was confirmed also by MALDI-MS. Among other changes in IgA glycosylation determined by lectin-based protein microarray were, for example, reduced galactosylation after supplementation with succinic acid and increase of both sialylation and galactosylation after supplementation with glutamine and feeding with mannose. The elucidation of mechanism of determined changes requires further investigation, but the described analytical approach represent effective platform for determination, screening and evaluation of glycosylation of therapeutic proteins.

Sensitive glycoprofiling of insulin-like growth factor receptors isolated from colon tissue of patients with colorectal carcinoma using lectin-based protein microarray.

Glycosylation of cell receptors influences their function and development of tumour induces changes in glycosylation. Cell growth depends on the activation of receptors which bind growth factors and the insulin-like growth factor (IGF) receptors are among the most important ones. Usually, only small quantities of isolated receptors are available thus there is a need of suitable assay to study receptors glycosylation. Therefore, we developed a lectin-based reverse-phase protein microarray method for screening the glycosylation pattern of receptors in picomolar (pM) concentrations. The method was applied to glycoprofile IGF1 and IGF2 receptors and the solubilised membrane proteins isolated from tumour and non-tumour colon tissue of patients with colorectal cancer. We found that common to both receptors was partial overlapping of the major glycan structures with those present in the entire glycome of membrane proteins. In contrast, receptors possess higher level of α2,3 sialic acid residues and lower level of tri-/tetra-antennary complex type N-glycans and terminal mannose in high-mannose structures. Increased levels of fucosylation and branched mannose structures were observed in both receptors derived from tumour tissue compared to non-tumour tissue. The described method enabling glycan analysis of receptors has a big application potential in e.g. biomarker research, biology and diagnostics.

Glycoanalysis of the placental membrane glycoproteins throughout placental development.

Structural changes of glycans are observed in different (patho)physiological conditions. Human placental membrane (glyco)proteins were isolated from the first and third trimester placentas of mothers at different ages. By using lectin microarray, we demonstrated that the placental membrane N-glycome contains several N-glycan groups: high mannose, asialylated and sialylated biantennary moieties, bisected, core fucosylated, fucosylated at other positions (bearing terminal and/or antennary Fuc), α2-6 and α2-3 sialylated structures. Employing MALDI-TOF MS enabled identification of over sixty different N-glycan structures in all samples, with 17 moieties exceeding the relative abundance of 2%. The major MS peaks originated from: 1) biantennary complex type N-glycan with a bisecting GlcNAc residue and 2) a core Fuc paucimannosidic and high mannose type structures M3-M9. Age of mothers and the stage of placental development affected N-glycome. The work presented in this article is the first comprehensive mass spectrometric study of the N-glycome of human placental membrane proteins. Our results may be seen as the baseline which can serve for future MALDI MS profiling of the placental membrane N-glycome in different pathophysiological conditions.