Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway

Background

Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses.

Results

The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Le^x and H type 2 antennae in sialylated complex-type N-glycans.

Conclusion

The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.     

Mass spectrometric approach for screening modifications of total serum N-glycome in human diseases: application to cirrhosis

Congenital and acquired modifications of glycosylation in diseases are a rapidly growing field that demonstrates the importance of glycosylation in human biology. Unfortunately, in clinical biochemistry, very few tests are available to explore oligosaccharide metabolism on a large scale. Such an assay needs to be of high throughput, rapid, and preferentially noninvasive. In the present study, we describe a method to analyze qualitative variations of N-glycosylation of human serum proteins. The method is based on direct release of N-linked oligosaccharides from patient serum samples, a single-step purification, and a matrix-assisted laser desorption ionization time of flight mass spectrometric analysis. A complementary structural study of the released oligosaccharides was achieved by enzymatic digestions, linkage analysis, and electrospray ionization ion trap mass spectrometry (ESI-IT-MS) of the permethylated N-glycome. A total of 26 oligosaccharide structures were individualized, their presence in human serum being the result of the combination of the biosynthesis and catabolic pathways. Application of the protocol to the serum of patients with cirrhosis demonstrates the ability of this assay to identify acquired modifications of glycosylation. Furthermore, we have analyzed the N-glycans and showed the increase in bisecting N-acetylglucosamine residue, core fucosylation, and the presence of an important population of neutral oligosaccharides. The study of total serum N-glycome modifications is a preliminary for the discovery of new noninvasive diagnostic or prognostic biomarkers resulting from the variations of the N-glycan metabolism during diseases.     

Analysis of N-glycan in serum glycoproteins from db/db mice and humans with type 2 diabetes

Glycosylation has an important role in regulating properties of proteins and is associated with many diseases. To examine the alteration of serum N-glycans in type 2 diabetes, we used the db/db mouse model. Serum N-glycans were fluorescence labeled and applied to HPLC. There were reproducible differences in N-glycan profiles between the db/db mouse model and the db/+ control. The structures of the oligosaccharides, which had changed in their amounts, were analyzed by a two-dimensional mapping method, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and exoglycosidase digestion. Those analyses revealed an increase in the N-glycans possessing alpha1,6-fucose in the serum of db/db mice. The level of alpha1,6-fucosyltransferase mRNA was increased in the liver of the db/db mice. The ratio of a biantennary N-glycan with alpha1,6-fucose to that without alpha1,6-fucose in the liver tissue of the db/db mouse was increased relative to the db/+ control. Next, we analyzed the serum N-glycan profile in human subjects with type 2 diabetes and found an increased amount of a biantennary N-glycan that had an alpha1,6-fucose with a bisecting N-acetylglucosamine. In conclusion, the increase in alpha1,6-fucosylation is a striking change in the serum N-glycans of the db/db mice, whereas the change in the fucosylation in humans with type 2 diabetes was small, albeit statistically significant. It is likely that the change is caused, at least partially, by the increase in the alpha1,6-fucosyltransferase mRNA level in the liver. The increased alpha1,6-fucosylation may affect protein properties associated with the pathophysiology of type 2 diabetes.     

Comparison of the human and bovine milk N-glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry

The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.     

N-linked glycan structures and their expressions change in the blood sera of ovarian cancer patients

Glycosylated proteins play important roles in a broad spectrum of biochemical and biological processes, and prior reports have suggested that changes in protein glycosylation occur during cancer initiation and progression. Ovarian cancer (OC) is a fatal malignancy, most commonly diagnosed after the development of metastases. Therefore, early detection of OC is key to improving survival. To this end, specific changes of the serum glycome have been proposed as possible biomarkers for different types of cancers. In this study, we extend this concept to OC. To characterize differences in total N-glycan levels, serum samples provided by 20 healthy control women were compared to those acquired from patients diagnosed with late-stage recurrent OC who were enrolled in an experimental treatment trial prior to receiving therapy (N=19) and one month later, prior to the second treatment cycle (N=11). Additionally, analyses of the N-glycans associated with IgG and characterization of the relative abundance levels of core vs outer-arm fucosylation were also performed. The N-linked glycomic profiles revealed increased abundances of tri- and tetra-branched structures with varying degrees of sialylation and fucosylation and an apparent decrease in the levels of "bisecting" glycans in OC samples compared to controls. Increased levels of a-galactosylation structures were observed on N-linked glycans derived from IgG, which were independent of the presence of fucose residues. Elevated levels of outer-arm fucosylation were also identified in the OC samples. These results allowed the control samples to be distinguished from the baseline ovarian cancer patients prior to receiving the experimental treatment. In some cases, the pre-treatment samples could be distinguished from the post-experimental treatment samples, as many of those patients showed a further progression of the disease.     

HPLC analysis of discrete haptoglobin isoform N-linked oligosaccharides following 2D-PAGE isolation

Glycosylation is a common but variable modification that regulates glycoprotein structure and function. We combined small format 2D-PAGE with HPLC to analyse discrete human haptoglobin isoform N-glycans. Seven major and several minor haptoglobin isoforms were detected by 2D-PAGE. N-Glycans released from Coomassie-stained gel spots using PNGase were labeled at their reducing termini with 2-aminobenzamide. HPLC analysis of selected major isoform N-glycans indicated that sialic acid composition determined their separation by isoelectric focussing. N-Glycans from two doublets of quantitatively minor isoforms were also analysed. Although separation of each pair of doublets was influenced by sialylation, individual spots within each doublet contained identical N-glycans. Thus, heterogeneity in minor haptoglobin isoforms was due to modifications distinct from N-glycan structure. These studies describe a simple method for analysing low abundance protein N-glycans and provide details of discrete haptoglobin isoform N-glycan structures which will be useful in proteomic analysis of human plasma samples.     

Fucosylated haptoglobin is a novel marker for pancreatic cancer: detailed analyses of oligosaccharide structures

Changes in oligosaccharide structures have been reported in certain types of malignant transformation and thus can be used as tumor markers in certain types of cancer. In the case of pancreatic cancer (PC) cell lines, a variety of fucosylated proteins are secreted into the conditioned media. To identify fucosylated proteins in the sera of patients with PC, we performed Western blot analysis using Aleuria Aurantia Lectin (AAL), which is specific for fucosylated structures. An approximately 40 kD protein was found to be highly fucosylated in PC and N-terminal analysis revealed that it was the beta chain of haptoglobin. While the appearance of fucosylated haptoglobin has been reported in other diseases such as hepatocellular carcinoma, liver cirrhosis, gastric cancer, and colorectal cancer, the incidence was significantly higher in the case of PC. Fucosylated haptoglobin was observed more frequently at the advanced stage of PC and disappeared after operation. Haptoglobin has four sites of N-glycans and site-directed oligosaccharide analysis involving MS was performed. Site-specific increases in fucosylation of bi-antennary glycans of sites 2 and 4, and of tri-antennary glycans of all sites were observed in PC, compared to in normal volunteers and chronic pancreatitis. Therefore, increases in fucosylation seem to be not due to inflammation, but cancer itself. Coculturing of a human hepatoma cell line, Hep3B, with PC cells-induced production of fucosylated haptoglobin, suggesting that PC produces a factor that induces the production of fucosylated haptoglobin. On clinical investigation of 100 cases of colorectal cancer, cases in which it was located near the liver showed a higher positive rate of fucosylated haptoglobin, suggesting that the location of the cancer might also be an important factor for fucosylated haptoglobin if cancer tissues produce such inducible factors. Thus, fucosylated haptoglobin could become a novel tumor marker for PC and complicated mechanisms would be involved in its production.     

Alterations in the serum glycome due to metastatic prostate cancer

Glycomic profiles derived from human blood sera of 10 healthy males were compared to those from 24 prostate cancer patients. The profiles were acquired using MALDI-MS of permethylated N-glycans released from 10-microL sample aliquots. Quantitative permethylation was attained using solid-phase permethylation. Principal component analysis of the glycomic profiles revealed significant differences among the two sets, allowing their distinct clustering. The first principal component distinguished the 24 prostate cancer patients from the healthy individuals. It was determined that fucosylation of glycan structures is generally higher in cancer samples (ANOVA test p-value of 0.0006). Although more than 50 N-glycan structures were determined, 12 glycan structures, of which six were fucosylated, were significantly different between the two sample sets. Significant differences were confirmed through two independent statistical tests (ANOVA and ROC analyses). Ten of these structures had significantly higher relative intensities in the case of the cancer samples, while the other two were less abundant in the cancer samples. All 12 structures were statistically significant, as suggested by their very low ANOVA scores (<0.001) and ROC analysis, with area under the curve values close to 1 or 0. Accordingly, these structures can be considered as cancer-specific glycans and potential prostate cancer biomarkers. Therefore, serum glycomic profiling appears worthy of further investigation to define its role in cancer early detection and prognostication.     

N-linked glycosylation profiling of pancreatic cancer serum using capillary liquid phase separation coupled with mass spectrometric analysis

Glycoproteins play important roles in various biological processes including intracellular transport, cell recognition, and cell-cell interactions. The change of the cellular glycosylation profile may have profound effects on cellular homeostasis and malignancy. Therefore, we have developed a sensitive screening approach for the comprehensive analysis of N-glycans and glycosylation sites on human serum proteins. Using this approach, N-linked glycopeptides were extracted by double lectin affinity chromatography. The glycans were enzymatically cleaved from the peptides and then profiled using capillary hydrophilic interaction liquid chromatography coupled online with ESI-TOF MS. The structures of the separated glycans were determined by MALDI quadrupole ion-trap TOF mass spectrometry in both positive and negative modes. The glycosylation sites were elucidated by sequencing of PNGase F modified glycopeptides using nanoRP-LC-ESI-MS/MS. Alterations of glycosylation were analyzed by comparing oligosaccharide expression of serum glycoproteins at different disease stages. The efficiency of this method was demonstrated by the analysis of pancreatic cancer serum compared to normal serum. Ninety-two individual glycosylation sites and 202 glycan peaks with 105 unique carbohydrate structures were identified from approximately 25 mug glycopeptides. Forty-four oligosaccharides were found to be distinct in the pancreatic cancer serum. Increased branching of N-linked oligosaccharides and increased fucosylation and sialylation were observed in samples from patients with pancreatic cancer. The methodology described in this study may elucidate novel, cancer-specific oligosaccharides and glycosylation sites, some of which may have utility as useful biomarkers of cancer.     

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.     

Comprehensive serum N-glycan profiling identifies a biomarker panel for early diagnosis of non-small-cell lung cancer

Aberrant serum N-glycan profiles have been observed in multiple cancers including non-small-cell lung cancer (NSCLC), yet the potential of N-glycans in the early diagnosis of NSCLC remains to be determined. In this study, serum N-glycan profiles of 275 NSCLC patients and 309 healthy controls were characterized by MALDI-TOF-MS. The levels of serum N-glycans and N-glycosylation patterns were compared between NSCLC and control groups. In addition, a panel of N-glycan biomarkers for NSCLC diagnosis was established and validated using machine learning algorithms. As a result, a total of 54 N-glycan structures were identified in human serum. Compared with healthy controls, 29 serum N-glycans were increased or decreased in NSCLC patients. N-glycan abundance in different histological types or clinical stages of NSCLC presented differentiated changes. Furthermore, an optimal biomarker panel of eight N-glycans was constructed based on logistic regression, with an AUC of 0.86 in the validation set. Notably, this model also showed a desirable capacity in distinguishing early-stage patients from healthy controls (AUC = 0.88). In conclusion, our work highlights the abnormal N-glycan profiles in NSCLC and provides supports potential application of N-glycan biomarker panel in clinical NSCLC detection.     

Site-specific and linkage analyses of fucosylated <Emphasis Type="Italic">N</Emphasis>-glycans on haptoglobin in sera of patients with various types of cancer: possible implication for the differential diagnosis of cancer

Fucosylation is an important type of glycosylation involved in cancer, and fucosylated proteins could be employed as cancer biomarkers. Previously, we reported that fucosylated N-glycans on haptoglobin in the sera of patients with pancreatic cancer were increased by lectin-ELISA and mass spectrometry analyses. However, an increase in fucosylated haptoglobin has been reported in various types of cancer. To ascertain if characteristic fucosylation is observed in each cancer type, we undertook site-specific analyses of N-glycans on haptoglobin in the sera of patients with five types of operable gastroenterological cancer (esophageal, gastric, colon, gallbladder, pancreatic), a non-gastroenterological cancer (prostate cancer) and normal controls using ODS column LC-ESI MS. Haptoglobin has four potential glycosylation sites (Asn184, Asn207, Asn211, Asn241). In all cancer samples, monofucosylated N-glycans were significantly increased at all glycosylation sites. Moreover, difucosylated N-glycans were detected at Asn 184, Asn207 and Asn241 only in cancer samples. Remarkable differences in N-glycan structure among cancer types were not observed. We next analyzed N-glycan alditols released from haptoglobin using graphitized carbon column LC-ESI MS to identify the linkage of fucosylation. Lewis-type and core-type fucosylated N-glycans were increased in gastroenterological cancer samples, but only core-type fucosylated N-glycan was relatively increased in prostate cancer samples. In metastatic prostate cancer, Lewis-type fucosylated N-glycan was also increased. These data suggest that the original tissue/cell producing fucosylated haptoglobin is different in each cancer type and linkage of fucosylation might be a clue of primary lesion, thereby enabling a differential diagnosis between gastroenterological cancers and non-gastroenterological cancers.     

Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits

The large-scale production of recombinant biopharmaceutical glycoproteins in the milk of transgenic animals is becoming more widespread. However, in comparison with bacterial, plant cell, or cell culture production systems, little is known about the glycosylation machinery of the mammary gland, and hence on the glycosylation of recombinant glycoproteins produced in transgenic animals. Here the influence is presented of several lactation parameters on the N-glycosylation of recombinant C1 inhibitor (rhC1INH), a human serum glycoprotein, expressed in the milk of transgenic rabbits. Enzymatically released N-glycans of series of rhC1INH samples were fluorescently labeled and fractionated by HPLC. The major N-glycan structures on rhC1INH of pooled rabbit milk were similar to those on native human C1 inhibitor and recombinant human C1 inhibitor produced in transgenic mouse milk, with only the degree of sialylation and core fucosylation being lower. Analyses of individual animals furthermore showed slight interindividual differences; a decrease in the extent of sialylation, core fucosylation, and oligomannose-type glycosylation with the progress of lactation; and a positive correlation between expression level and oligomannose-type N-glycan content. However, when large quantities of rhC1INH were isolated for preclinical and clinical studies, highly consistent N-linked glycan profiles and monosaccharide compositions were found.     

N-linked oligosaccharides of vomeromodulin, a putative pheromone transporter in rat

Vomeromodulin, a putative pheromone transporter of the rat vomeronasal organ, was isolated by lectin chromatography, purified, and subjected to a mass spectrometric (MS) system of glycan structural determination. Through a combination of exoglycosidase treatments and measurements by matrix-assisted laser desorption/ionization MS, the N-glycans of vomeromodulin were identified as mainly sialylated and fucosylated biantennary structures. The microheterogeneity of N-glycan structures was also due to the presence of galactose residues with different types of linkages.     

Identification of an inducible factor secreted by pancreatic cancer cell lines that stimulates the production of fucosylated haptoglobin in hepatoma cells

Fucosylation is one of the most important oligosaccharide modifications and is involved in cancer and inflammation. Recently, fucosylated haptoglobin was identified as a possible tumor marker for pancreatic cancer. The molecular mechanism underlying increases in fucosylated haptoglobin in sera of patients with pancreatic cancer seems to be complicated. Our previous study [N. Okuyama, Y. Ide, M. Nakano, T. Nakagawa, K. Yamanaka, K. Moriwaki, K. Murata, H. Ohigashi, S. Yokoyama, H. Eguchi, O. Ishikawa, T. Ito, M. Kato, A. Kasahara, S. Kawano, J. Gu, N. Taniguchi, E. Miyoshi, Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation, Int. J. Cancer 118 (11) (2006) 2803-2808] demonstrated that pancreatic cancer cells secrete a factor, which induces the production of haptoglobin in hepatoma cells. In the present study, we found that interleukin 6 (IL6) expressed in pancreatic cancer is a factor that induces the haptoglobin production, using a neutralizing antibody for IL6. Real-time PCR analyses revealed the up-regulation of fucosylation regulatory genes after IL6 treatment, resulting increases in fucosylated haptoglobin being revealed by a lectin ELISA. This pathway could be one of the possible mechanisms underlying increases in haptoglobin in sera of patients with pancreatic cancer.     

Characterization of the N-linked glycans of Giardia intestinalis

This article reports the first rigorous evidence for the existence of N-glycans in Giardia intestinalis, a parasite that is a widespread human pathogen, being a major cause of enteric disease in the world. Excreted/secreted molecules of G. intestinalis are known to stimulate the immune system. Structural strategies based on MALDI and electrospray mass spectrometry were employed to examine the excreted/secreted molecules for their N-glycan content. These revealed that the major oligosaccharides released by peptide N-glycosidase F are complex-type structures and correspond to bi-, and triantennary structures without core (alpha1,6) fucosylation. The major nonreducing epitopes in these complex-type glycans are: Galbeta1-4GlcNAc (LacNAc) and NeuAc alpha2-6Galbeta1-4GlcNAc (sialylated LacNAc).     

Unique Asn-linked oligosaccharides of the human pathogen Entamoeba histolytica

N-Glycans of Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, are of great interest for multiple reasons. E. histolytica makes an unusual truncated N-glycan precursor (Man(5)GlcNAc(2)), has few nucleotide sugar transporters, and has a surface that is capped by the lectin concanavalin A. Here, biochemical and mass spectrometric methods were used to examine N-glycan biosynthesis and the final N-glycans of E. histolytica with the following conclusions. Unprocessed Man(5)GlcNAc(2), which is the most abundant E. histolytica N-glycan, is aggregated into caps on the surface of E. histolytica by the N-glycan-specific, anti-retroviral lectin cyanovirin-N. Glc(1)Man(5)GlcNAc(2), which is made by a UDP-Glc: glycoprotein glucosyltransferase that is part of a conserved N-glycan-dependent endoplasmic reticulum quality control system for protein folding, is also present in mature N-glycans. A swainsonine-sensitive alpha-mannosidase trims some N-glycans to biantennary Man(3)GlcNAc(2). Complex N-glycans of E. histolytica are made by the addition of alpha1,2-linked Gal to both arms of small oligomannose glycans, and Gal residues are capped by one or more Glc. In summary, E. histolytica N-glycans include unprocessed Man(5)GlcNAc(2), which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc.     

Enhancement of ovarian tumor classification by improved reproducibility in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of serum glycans

The serum N-glycome is a promising source of biomarker discovery. Matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry (MS) profiling of serum N-glycans was attempted for differentiating borderline ovarian tumor from benign cases, for which a low data spread is essential. An experimental protocol using matrix-prespotted MALDI plates and fast vacuum drying of the loaded N-glycan samples was developed, thereby minimizing the intensity variations in the replicates to an average relative standard deviation (RSD) of 3.96% for the highest N-glycan peak (m/z 1485.53) of the Sigma–Aldrich serum standard. When applied to sera of ovarian tumors, this procedure exhibited an average RSD of 5.74% for m/z 1485.53 and of 7.28% for all MS peaks. This improved reproducibility combined with the OVA-Beyond^® screening software resulted in 75.1% and 79.4% correct classification for benign and borderline tumor samples, respectively, while the classification rates by the conventional ovarian tumor marker CA-125 were 54.4% and 53.1%, respectively. Both true positive rate and true negative rate fluctuated with small numbers of markers and converged as the number of markers increased. Cross-validations were performed in comparison with CA-125. These results suggest that our optimized process for MALDI–TOF MS of the serum glycome has a great potential for the screening of early stage ovarian cancer.     

N-glycosylation patterns of HSA/CD24 from different cell lines and brain homogenates: a comparison

N-glycans of the mouse glycoprotein HSA and its human analogue CD24 from lymphoblastoma, neuroblastoma and astrocytoma cell lines as well as from mouse brain homogenate were analysed and compared to each other and to the N-glycosylation pattern of total glycoproteins from mouse and human brain. The N-glycans were released from PVDF-blotted HSA or CD24 and separated on Carbograph SPE into neutral and acid glycans. The naturally neutral glycan fraction and the fraction of glycans rendered neutral after neuraminidase treatment were analysed without further purification by MALDI-MS. In each fraction, about 25 molecular ions with an intensity >10% of the base peak were identified which corresponded to glycans with distinct isobaric monosaccharide compositions. Comparison of the neutral and desialylated glycans revealed some similarities between the samples analysed, but also clear differences. HSA and CD24 from all cell lines express almost no neutral N-glycans with two or more fucose in contrast to brain HSA and glycoproteins from mouse and human brain. The lack of extensive fucosylation was also observed for desialylated glycans of HSA and CD24 from all cell lines analysed except for CD24 from a human neuroblastoma cell line which exhibits like total human and mouse brain glycoproteins a large variety of highly fucosylated, higher branched N-glycans. HSA from mouse brain carries in addition desialylated non-fucosylated glycans of high abundance which were detected, if at all, only at low intensity in all other samples analysed suggesting that they may be implicated in specific functions of mouse brain HSA. Therefore, a rapid assessment of similarities or differences between glycosylation patterns of a glycoprotein isolated from different sources is possible using methods as described here.     

The mapping by high-pH anion-exchange chromatography with pulsed amperometric detection and capillary electrophoresis of the carbohydrate moieties of human plasma alpha 1-acid glycoprotein.

The reducing oligosaccharides released from alpha 1-acid glycoprotein (AGP) by conventional hydrazinolysis have been analyzed by two different mapping techniques, using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and capillary electrophoresis (CE) with uv detection at 190 nm. The CE measurements proved about 4000 times more sensitive than the measurements by HPAE-PAD. The N-glycan pool was fractionated by Mono Q anion-exchange chromatography, and individual fractions so obtained were desialylated using Vibrio cholerae neuraminidase. The resulting asialo-N-glycans were further analyzed by HPAE-PAD, revealing 2 major, 4 intermediate, and 4 small peaks and at least 3 spikes, which counted for at least 13 different asialo-N-glycans. The carbohydrate structures were tentatively assigned by comparison of the Mono Q-separated N-glycans with the known AGP carbohydrate structures and known structures contained in a mapping database that allows structural assignment of N-glycans by mere comparison of retention times. In addition to the hitherto known AGP carbohydrate structures, we have tentatively identified a number of sulfated N-glycans that are currently being analyzed in more detail. We have also compared the glycan pools recovered from AGP using hydrazinolysis and glycopeptidase F (PNGase F). Approximately 40 distinct peaks could be detected in the hydrazinolysis-derived N-glycan pool by either technique (HPAE-PAD and CE), while about 30 distinct peaks were detected in the N-glycan pool derived by PNGase F digestion of the tryptic AGP digest of the same batch of AGP. These differences were attributed to an increased desialylation (approximately 3 mol%) during hydrazinolysis, based on the detection by HPAE-PAD and CE of free sialic acid and monosialylated oligosaccharides in the glycan pool derived by conventional hydrazinolysis. The integrity of the N-glycans' chitobiose core was examined by 500-MHz 1H NMR spectoscopy. The hydrazinolysis procedure could be optimized such that the hydrazinolysis-derived N-glycan pool was chromatographically essentially identical to the PNGase F-derived N-glycan pool. Hydrazinolysis proved best, with practically no loss of N-acetlylneuraminic acid and the closest resemblance to the PNGase F-derived N-glycan pool, using an automated apparatus. Notably, it was recognized that, in our hands, PNGase F digestion in the presence of sodium dodecyl sulfate resulted in partial desialylation of the liberated N-glycans.