Analytical Investigation of the Profile of Human Chorionic Gonadotropin in Highly Purified Human Menopausal Gonadotrophin Preparations.

Highly purified human menopausal gonadotropin (HP-hMG [Menopur®, Ferring Pharmaceuticals, Saint-Prex, Switzerland]) contains a 1:1 ratio of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This analysis aimed to assess gonadotropin (FSH, LH and hCG) abundance in HP-hMG and clarify the source of hCG by assessing the presence of sulfated glycans, which are diagnostic for pituitary hCG forms due to their distinct glycosylation patterns. Additionally, the purity of each sample, their specific components, and their oxidation levels were assessed. HP-hMG samples (three of Menopur® and two of Menogon® Ferring Pharmaceuticals, Saint-Prex, Switzerland) were included in the current analyses. Brevactid® (urinary hCG; Ferring Pharmaceuticals, Saint-Prex, Switzerland) and Ovidrel® (recombinant hCG; Merck KGaA, Darmstadt, Germany) were used as control samples. Glycopeptide mapping and analysis of impurities were carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oxidation was assessed through reducing peptide mapping using LC-MS/MS. The FSH and LH in the HP-hMG samples showed sulfated glycans, while no signals of sulfated glycopeptides were detected on any site of the beta subunit of hCG. HP-hMG test samples presented the same hCG glycan distribution as the control sample (placental hCG, Brevactid®) extracted from the urine of pregnant women, suggesting a non-pituitary source of hCG. Protein impurities were estimated to constitute approximately 20-30% of the entire HP-hMG protein content in the test samples. More than 200 non-gonadotropin proteins were identified in the HP-hMG test samples, of which several were involved in embryonic development or pregnancy. The alpha subunit of the tested samples was strongly oxidized, with a relative abundance of 20% of the total gonadotropin content. Without taking into account all the protein impurities, the beta subunit of LH was detected only in traces (0.9-1.2%) in all tested HP-HMG samples, confirming the data obtained by intact molecule analysis, while high levels of beta hCG (18-47%) were observed. Advanced molecular analysis of HP-hMG indicates a primarily placental origin of hCG, as evidenced by the absence of hCG sulfated glycans and the predominance of placental non-sulfated hCG in LH activity. The analysis revealed 20-30% of protein impurities and a significant presence of oxidized forms in the HP-hMG samples. These findings are critical for understanding the quality, safety, and clinical profile of HP-hMG.

Identification of ß1-3 galactosylglucose-core free-glycans in human urine.

We previously reported the precise structure of acidic free-glycans in human urine. In the present study, structural analysis of neutral free-glycans in urine was performed in fine detail. Urine samples were collected from 21 healthy volunteers and free-glycans extracted from the creatinine-adjusted urine and then fluorescently labeled with 2-aminopyridine. Neutral glycan profiling was achieved by a combination of high-performance liquid chromatography, mass spectrometry, enzymatic digestion, and periodate cleavage. A total of 79 glycans were identified. Because the ABO-blood group antigen containing urinary neutral glycans are major components, profiling patterns were similar between individuals of the same ABO-group. The neutral glycans were composed of lactose-core (Galß1-4Glc) glycans, type-II N-acetyllactosamine-core (GlcNAcß1-4Glc) glycans, hexose oligomers, N-glycans and to our surprise ß1-3 galactosylglucose-core (Galß1-3Glc) glycans. Although glycans with a ß1-3 galactosylglucose-core were identified as major components in urine, comprising structurally simple isomers of a lactose-core, the core structure has not previously been reported. The major ß1-3 galactosylglucose-core glycans were Fucα1-2Galß1-3(Fucα1-4)Glc, GalNAcα1-3(Fucα1-2)Galß1-3(Fucα1-4)Glc and Galα1-3(Fucα1-2)Galß1-3(Fucα1-4)Glc, corresponding to H-, A-, and B-blood group antigens, respectively. Three lactosamine extended ß1-3 galactosylglucose-core glycans were also detected as minor components. Elucidating the biosynthesis of ß1-3 galactosylglucose will be crucial for understanding the in vivo function of these glycans.

An in-depth Comparison of the Pediatric and Adult Urinary N-glycomes.

We performed an in-depth characterization and comparison of the pediatric and adult urinary glycomes using a nanoLC-MS/MS based glycomics method, which included normal healthy pediatric (1-10 years, n = 21) and adult (21-50 years, n = 22) individuals. A total of 116 N-glycan compositions were identified, and 46 of them could be reproducibly quantified. We performed quantitative comparisons of the 46 glycan compositions between different age and sex groups. The results showed significant quantitative changes between the pediatric and adult cohorts. The pediatric urinary N-glycome was found to contain a higher level of high-mannose (HM), asialylated/afucosylated glycans (excluding HM), neutral fucosylated and agalactosylated glycans, and a lower level of trisialylated glycans compared with the adult. We further analyzed gender-associated glycan changes in the pediatric and adult group, respectively. In the pediatric group, there was almost no difference of glycan levels between males and females. In adult, the majority of glycans were more abundant in males than females, except the high-mannose and tetrasialylated glycans. These findings highlight the importance to consider age-matching and adult sex-matching for urinary glycan studies. The identified normal pediatric and adult urinary glycomes can serve as a baseline reference for comparisons to other disease states affected by glycosylation.

Magnetic porous carbon-dependent platform for the determination of N-glycans from urine exosomes.

A magnetic porous carbon-dependent platform is established to separate and determine N-glycans from urine exosomes of healthy people and patients with gastric cancer. The results of the comparison reveal that 6 N-glycans shared by the two groups are downregulated, most of which present core fucose or bisecting N-acetylglucosamine (GlcNAc) type. In addition, five shared N-glycans including two of sialic acid type are upregulated. These obvious differences indicate the close relationship between glycans and gastric cancer thus permitting early diagnosis. A magnetic porous carbon material (FeMPC) from MIL-101(Fe) was employed to separate and analyze N-glycans from urine exosomes of healthy people and patients with gastric cancer.

Glycans, Glycosite, and Intact Glycopeptide Analysis of N-Linked Glycoproteins Using Liquid Handling Systems.

Aberrant glycosylation has been shown to associate with disease progression, and with glycoproteins representing the major protein component of biological fluids this makes them attractive targets for disease monitoring. Leveraging glycoproteomic analysis via mass spectrometry (MS) could provide the insight into the altered glycosylation patterns that relate to disease progression. However, investigation of large sample cohorts requires rapid, efficient, and highly reproducible sample preparation. To address the limitation, we developed a high-throughput method for characterizing glycans, glycosites, and intact glycopeptides (IGPs) derived from N-linked glycoproteins. We combined disparate peptide enrichment strategies (i.e., hydrophilic and hydrophobic) and a liquid handling platform allowing for a high throughput and rapid enrichment of IGP in a 96-well plate format. The C18/MAX-Tip workflow reduced sample processing time and facilitated the selective enrichment of IGPs from complex samples. Furthermore, our approach enabled the analysis of deglycosylated peptides and glycans from enriched IGPs following PNGase F digest. Following development and optimization of the C18/MAX-Tip methodology using the standard glycoprotein, fetuin, we investigated normal urine samples to obtain N-linked glycoprotein information. Together, our method enables a high-throughput enrichment of glycan, glycosites, and IGPs from biological samples.

N-Linked Glycosylation and Near-Infrared Spectroscopy in the Diagnosis of Prostate Cancer.

BACKGROUND: Performing a prostate biopsy is the most robust and reliable way to diagnose prostate cancer (PCa), and to determine the disease grading. As little to no biochemical markers for prostate tissue exist, we explored the possibilities of tissue N-glycosylation and near-infrared spectroscopy (NIR) in PCa diagnosis. METHODS: Tissue specimens from 100 patients (benign prostate hyperplasia (BPH), n = 50; and PCa, n = 50) were obtained. The fresh-frozen tissue was dispersed and a tissue N-glycosylation profile was determined. Consequently, the formalin-fixed paraffin-embedded slides were analyzed using NIR spectroscopy. A comparison was made between the benign and malignant tissue, and between the various Gleason scores. RESULTS: A difference was observed for the tissue of N-glycosylation between the benign and malignant tissue. These differences were located in the fycosylation ratios and the total amount of bi- and tetra-antennary structures (all p < 0.0001). These differences were also present between various Gleason scores. In addition, the NIR spectra revealed changes between the benign and malignant tissue in several regions. Moreover, spectral ranges of 1055⁻1065 nm and 1450⁻1460 nm were significantly different between the Gleason scores (p = 0.0042 and p = 0.0195). CONCLUSIONS: We have demonstrated biochemical changes in the N-glycan profile of prostate tissue, which allows for the distinction between malignant and benign tissue, as well as between various Gleason scores. These changes can be correlated to the changes observed in the NIR spectra. This could possibly further improve the histological assessment of PCa diagnosis, although further method validation is needed.

Absorption Study of Mozuku Fucoidan in Japanese Volunteers.

We performed an oral administration study of fucoidan in 396 Japanese volunteers and investigated significant factors concerning the absorption of fucoidan. Urine samples were collected at 0, 3, 6, and 9 h after ingestion of 3 g of fucoidan. Fucoidan was detected in urine after ingestion in 385 out of 396 subjects. The maximum value (mean ± standard deviation (SD)) of urinary fucoidan was 332.3 ± 357.6 µg/gCr in subjects living in Okinawa prefecture, compared with 240.1 ± 302.4 µg/gCr in subjects living outside Okinawa. Compared with the estimated urinary excretion of fucoidan by place of residence, those of subjects living in Okinawa prefecture were significantly higher than those living outside Okinawa prefecture (p < 0.01). In addition, subjects living in Okinawa prefecture consumed significantly greater amounts of mozuku compared with those living outside Okinawa prefecture (p < 0.01). Multiple regression analysis showed that having Okinawa prefecture as a place of residence was a significant factor (p < 0.01) contributing to the estimated urinary excretion of fucoidan. Because the habit of eating mozuku was significantly higher (p < 0.01) in subjects living in Okinawa prefecture than in those living outside Okinawa prefecture, the habit of eating mozuku was speculated to be a factor in the absorption of fucoidan.

Comparative Assessment of Glycosylation of a Recombinant Human FSH and a Highly Purified FSH Extracted from Human Urine.

Glycosylation is an important PTM and is critical for the manufacture and efficacy of therapeutic glycoproteins. Glycan significantly influences the biological properties of human follicle-stimulating hormone (hFSH). Using a glycoproteomic strategy, this study compared the glycosylation of a putative highly purified FSH (uhFSH) obtained from human urine with that of a recombinant human FSH (rhFSH) obtained from Chinese hamster ovary (CHO) cells. Intact and subunit masses, N-glycans, N-glycosylation sites, and intact N- and O-glycopeptides were analyzed and compared by mass spectrometry. Classic and complementary analytical methods, including SDS-PAGE, isoelectric focusing, and the Steelman-Pohley bioassay were also employed to compare their intact molecular weights, charge variants, and specific activities. Results showed that highly sialylated, branched, and macro-heterogeneity glycans are predominant in the uhFSH compared with those in rhFSH. The O-glycopeptides of both hFSHs, which have not been described previously, were characterized herein. A high degree of heterogeneity was observed in the N-glycopeptides of both hFSHs. The differences in glycosylation provide useful information in elucidating and in further investigation the critical glycan structures of hFSH.

SweetSEQer, simple de novo filtering and annotation of glycoconjugate mass spectra.

The past 15 years have seen significant progress in LC-MS/MS peptide sequencing, including the advent of successful de novo and database search methods; however, analysis of glycopeptide and, more generally, glycoconjugate spectra remains a much more open problem, and much annotation is still performed manually. This is partly because glycans, unlike peptides, need not be linear chains and are instead described by trees. In this study, we introduce SweetSEQer, an extremely simple open source tool for identifying potential glycopeptide MS/MS spectra. We evaluate SweetSEQer on manually curated glycoconjugate spectra and on negative controls, and we demonstrate high quality filtering that can be easily improved for specific applications. We also demonstrate a high overlap between peaks annotated by experts and peaks annotated by SweetSEQer, as well as demonstrate inferred glycan graphs consistent with canonical glycan tree motifs. This study presents a novel tool for annotating spectra and producing glycan graphs from LC-MS/MS spectra. The tool is evaluated and shown to perform similarly to an expert on manually curated data.

Dual modifications strategy to quantify neutral and sialylated N-glycans simultaneously by MALDI-MS.

Differences in ionization efficiency among neutral and sialylated glycans prevent direct quantitative comparison by their respective mass spectrometric signals. To overcome this challenge, we developed an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), to quantitatively compare neutral and sialylated glycans simultaneously by MALDI-MS. Initially, two glycan samples to be compared undergo reductive amination with 2-aminobenzoic acid and 2-(13)[C6]-aminobenzoic acid, respectively. The different isotope-incorporated glycans are then combined and subjected to the methylamidation of the sialic acid residues in one mixture, homogenizing the ionization responses for all neutral and sialylated glycans. By this approach, the expression change of relevant glycans between two samples is proportional to the ratios of doublet signals with a static 6 Da mass difference in MALDI-MS and the change in relative abundance of any glycan within samples can also be determined. The strategy was chemically validated using well-characterized N-glycans from bovine fetuin and IgG from human serum. By comparing the N-glycomes from a first morning (AM) versus an afternoon (PM) urine sample obtained from a single donor, we further demonstrated the ability of DRAG strategy to measure subtle quantitative differences in numerous urinary N-glycans.

[Research progresses of pharmacokinetics of polysaccharides].

Pharmacokinetic analysis has attracted more and more attentions in the research field of bioactive natural product. However, there is limited study on the pharmacokinetics of polysaccharides. This paper focused on the research progresses of pharmacokinetics of polysaccharide, summarized the applications of chromatography, isotope labeling method, spectrophotometry, fluorospectrophotometry and biological assay in the analysis of polysaccharide pharmacokinetics, elucidated the behaviors of absorption, distribution, degradation and excretion of polysaccharide in experimental animals, and revealed the effects of physicochemical characteristic, administration dose and route on the pharmacokinetic properties of polysaccharide, which could be served as a reference for the related works.

Multiplex Determination of Glycan Profiles on Urinary Prostate-Specific Antigen by Quartz-Crystal Microbalance Combined with Surface-Enhanced Raman Scattering.

Prostate cancer remains a major health concern, with prostate-specific antigen (PSA) being a key biomarker for its detection and monitoring. However, PSA levels often fall into a "gray zone", where PSA levels are not clearly indicative of cancer, thus complicating early diagnosis and treatment decisions. Glycosylation profiles, which often differ between healthy and diseased cells, have emerged as potential biomarkers to enhance the specificity and sensitivity of cancer diagnosis in these ambiguous cases. We propose the integration of two complementary techniques, namely quartz-crystal microbalance with dissipation (QCM-D) and surface-enhanced Raman scattering (SERS) to study PSA glycan profiles. QCM-D offers real-time operation, PSA mass quantification, and label-free detection with high sensitivity, as well as enhanced specificity and reduced cross-reactivity when using nucleic acid aptamers as capture ligands. Complementary SERS sensing enables the determination of the glycosylation pattern on PSA, at low concentrations and without the drawbacks of photobleaching, thereby facilitating multiplexed glycosylation pattern analysis. This integrated setup could retrieve a data set comprising analyte concentrations and associated glycan profiles in relevant biological samples, which may eventually improve early disease detection and monitoring. Prostate-specific antigen (PSA), a glycoprotein secreted by prostate epithelial cells, serves as our proof-of-concept analyte. Our platform allows multiplex targeting of PSA multiplex glycosylation profiles of PSA at "gray zone" concentrations for prostate cancer diagnosis. We additionally show the use of SERS for glycan analysis in PSA secreted from prostate cancer cell lines after androgen-based treatment. Differences in PSA glycan profiles from resistant cell lines after androgen-based treatment may eventually improve cancer treatment.

Evaluation of a novel point-of-care cryptococcal antigen test on serum, plasma, and urine from patients with HIV-associated cryptococcal meningitis.

BACKGROUND: Many deaths from cryptococcal meningitis (CM) may be preventable through early diagnosis and treatment. An inexpensive point-of-care (POC) assay for use with urine or a drop of blood would facilitate early diagnosis of cryptococcal infection in resource-limited settings. We compared cryptococcal antigen (CRAG) concentrations in plasma, serum, and urine from patients with CM, using an antigen-capture assay for glucuronoxylomannan (GXM) and a novel POC dipstick test. METHODS: GXM concentrations were determined in paired serum, plasma, and urine from 62 patients with active or recent CM, using a quantitative sandwich enzyme-linked immunosorbent assay (ELISA). A dipstick lateral-flow assay developed using the same monoclonal antibodies for the sandwich ELISA was tested in parallel. Correlation coefficients were calculated using Spearman rank test. RESULTS: All patients had detectable GXM in serum, plasma, and urine using the quantitative ELISA. Comparison of paired serum and plasma showed identical results. There were strong correlations between GXM levels in serum/urine (r(s) = 0.86; P < .001) and plasma/urine (r(s) = 0.85; P < .001). Levels of GXM were 22-fold lower in urine than in serum/plasma. The dipstick test was positive in serum, plasma, and urine in 61 of 62 patients. Dipstick titers correlated strongly with ELISA. Correlations between the methods were 0.93 (P < .001) for serum, 0.94 (P < .001) for plasma, and 0.94 (P < .001) for urine. CONCLUSIONS: This novel dipstick test has the potential to markedly improve early diagnosis of CM in many settings, enabling testing of urine in patients presenting to health care facilities in which lumbar puncture, or even blood sampling, is not feasible.

Urinary glycan markers for disease.

Robust assays for the isolation and characterization of urinary FOS (free oligosaccharides) have been developed to screen patients for altered protein and/or lipid glycosylation. A FOS analysis can therefore identify potential biomarkers for hepatocellular carcinoma, since variations in glycosylation as a result of tumorigenecity should be detectable in the FOS of patients. HCC (hepatocellular carcinoma) accounts for 80-90% of all liver cancers. It occurs more often in men than women and occurs mostly in people 50-60 years old. The disease is more common in parts of Africa and Asia than in North or South America and Europe. Using a combination of solid-phase extraction techniques and affinity chromatography, followed by separation of urinary FOS by NP (normal phase)-HPLC and HIAX (hydrophilic interaction and anion-exchange)-HPLC, more than 200 different species have been identified in patient samples. The high incidence of small sialylated oligosaccharides in HCC patients suggests that pro-inflammatory markers may be detected as early indicators of disease progression. In addition, the methods developed here to isolate and analyse excreted glycoprotein- and glycosphingolipid-bound oligosaccharides have been used to characterize changes in metabolic processes that underlie a number of human genetic disorders. The ability to predict disease status in microlitre amounts of readily available non-invasive urine samples indicates that rapid methods for screening can be developed.

Clinically Viable Assay for Monitoring Uromodulin Glycosylation.

Uromodulin, also known as the Tamm-Horsfall protein or THP, is the most abundant protein excreted in human urine. It is associated with the progression of kidney diseases; therefore, changes in the glycosylation profile of this protein could serve as a potential biomarker for kidney health. The typical glycomics analysis approaches used to quantify uromodulin glycosylation involve time-consuming and tedious glycoprotein isolation and labeling steps, which limit their utility in clinical glycomics assays, where sample throughput is important. Herein, we introduce a radically simplified sample preparation workflow, with direct ESI-MS analysis, enabling the quantification of N-linked glycans that originate from uromodulin. The method omits any glycan labeling steps but includes steps to reduce the salt content of the samples, thereby minimizing ion suppression. The method is effective for quantifying subtle glycosylation differences of uromodulin samples derived from different biological states. As a proof of concept, glycosylation from samples that differ by pregnancy status were shown to be differentiable.

Development of a fucoidan-specific antibody and measurement of fucoidan in serum and urine by sandwich ELISA.

Fucoidan exhibits various biological properties. We raised a novel antibody against fucoidan extracted from Cladosiphon okamuranus and developed a sandwich ELISA method to measure fucoidan. The fucoidan antibody was specific and did not cross-react with other polysulfated polysaccharides. Fucoidan recovery from serum and urine by ELISA was 86-113%. Intra- and inter-assay CVs were 1.5-13.4%. Assay linearity was maintained after 3-fold dilution of each sample with phosphate-buffer saline (PBS). In the serum and urine of healthy volunteers (n=10), fucoidan was not detected before administration, and the levels markedly increased 6 and 9 h after oral administration. The molecular weight of the serum fucoidan determined by HPLC gel filtration remained unchanged, whereas that of urine fucoidan was significantly reduced. This is the first ELISA method of measuring serum and urine fucoidan levels after oral administration. The method is simple, reliable, and practical for the analysis of samples, especially urine samples.

Is the transformation of fucoidans in human body possible?

Fucoidans are a group of homo-and hetero-polysaccharides, which necessarily contains residues of sulfated α-L-fucose. Fucoidans are found only in brown algae. These polysaccharides exhibit a wide spectrum of biological activity and have a great therapeutic potential. Enzymes capable of catalyzing the degradation of fucoidans are absent in the mammalian enzyme system. The question arises: is the transformation of fucoidan in mammals, particularly in human possible? Studies in vivo (in situ) and in vitro have demonstrated that high molecular weight fucoidans are absorbed across rat intestinal epithelial cells, accumulated by liver macrophages, and characterized by low levels in blood and urine. Using the example of the Okinawa Prefecture (Japan) residents, it was shown that Cladosiphon okamuranus alga is digested and the fucoidan contained in this alga is absorbed in the human body.

Software platform for high-throughput glycomics.

Mass spectrometry (MS) is a key tool for structural analysis of oligosaccharides because of its high accuracy, sensitivity, and speed on one hand and because of the general and flexible protocols on the other. In glycomics projects the analysis of mass spectra is the speed determining step because, unlike in proteomics, software platforms for high-throughput glycan mass spectra interpretation are not fully automated and still depend on highly specialized knowledge. For the publicly available software, initial steps for manual MS data preprocessing are required mostly considering operations with glycan structures already stored in databases. In particular, monoisotopic peaks have to be manually determined or imported. In this contribution we describe our development of a platform for MS data evaluation in glycomics that demands only a low human intervention. The proposed platform named SysBioWare is constructed to allow import of the raw MS data to the spectrum browser and to perform isotopic grouping of detected peaks after de-noising and wavelet analysis. Monoisotopic m/z values render peak list association with the raw MS spectrum and allow compositional assignment according to the tuned building block library. This platform has been applied to human urine glycome as a potent tool for rapid assignment of already known or/and novel structures.

Increased inositol phosphoglycan P-type in the second trimester in pregnant women with type 2 and gestational diabetes mellitus.

A progressive insulin resistant state develops throughout human pregnancy. Inositol phosphoglycan P-type (P-IPG), a second messenger of insulin, was reported to negatively correlate with the degree of insulin resistance in non-pregnant diabetic subjects. Urinary levels of P-IPG were assessed in insulin resistant states during pregnancy such as gestational diabetes mellitus (GDM, n=44) and type 2 diabetes mellitus (type 2 DM, n=25) and in 69 normal pregnant women. Urinary levels of P-IPG were higher in GDM than controls with a positive trend of release throughout normal pregnancy (P<0.01). P-IPG excretion was higher in diabetic (GDM and type 2 DM) than in healthy women in the second trimester (P<0.05). A higher P-IPG urinary excretion occurs during the second trimester in pregnant women with clinically evident insulin resistance with a positive association with poor glycemic control.

Can glycans unveil the origin of glycoprotein hormones? - human chorionic gonadotrophin as an example -.

Doping with (glyco)protein hormones represent an extremely challenging, analytical problem as nearly all are constitutively present at low concentrations that fluctuate according to circadian or alternative periodical, or external stimuli. Thus the mere concentration in a biological sample is only resolutive when this surpasses extreme values. As the vast majority of these molecules are produced by recombinant DNA technology it is believed that the exogenous molecules could bear the signature of the host cell. In particular, these could comprise structural differences originated from co or post-translational differences. In this study we have employed both proteomics and glycomics strategies to compare recombinant and urinary human chorionic gonadotrophin in order to evaluate this hypothesis. As anticipated the recombinant hormone could be shown to contain N-glycolyl neuraminic acid, a sialic acid that cannot be produced by humans. Furthermore, differences were observed in the overall glycosylation, in particular the presence of abundant hybrid-type glycans that were much less pronounced in the recombinant species. These differences were determined to occur predominantly in the alpha-subunit for which antidoping strategies focussed on these elements could be used for both chorionic gonadotrophin and lutrophin as they share the same alpha-subunit.