Pine Brook, NJ, United States
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Nouso K.,Okayama University of Science | Amano M.,Field of Drug Discovery Research | Amano M.,Hokkaido University | Ito Y.M.,Hokkaido University | And 19 more authors.
Journal of Gastroenterology | Year: 2013

Background: Most of the glycan changes reported in cancers were based on the examinations of a small number of patients or particular proteins. The aim of this study was to determine the changes of the serum N-glycan profile comprehensively in a large number of pancreatic cancer patients and investigate its clinical utility. Methods: Glycan levels in the serum of 92 pancreatic cancer patients and 243 healthy volunteers (HLT) were examined by comprehensive quantitative high-throughput glycome analysis and were compared with clinical parameters. Results: Out of 66 glycans detected, 15 were differentially expressed in pancreatic cancer, and 10 out of the 15 glycans were significantly up-regulated in cases with distant metastasis. There was a clear increase in overall expression of serum glycans, especially highly-branched glycans with fucose moieties, in pancreatic cancer. Among these 15 glycans, a tri-antennary complex type glycan (m/z 3195) showed the highest area under the receiver operating characteristic curve (AUROC = 0.799) for the diagnosis of pancreatic cancer. The ratio of pairs of glycans on the same path of the biosynthesis pathway (m/z 3195/1914) was found to be significantly higher in pancreatic cancer than in HLT (median = 1.11 and 0.41, respectively; p < 0.0001, AUROC = 0.831). For this pair ratio, the hazard ratio for survival (2.60, 95 % CI = 1.44-4.79) was higher than that of any single glycan and 1-year survival of patients with a high and low ratio was 36.9 and 69.2 %, respectively, (p = 0.001). Conclusions: Comprehensive glycome analysis can be used to know the presence of pancreatic cancer, distant metastasis, and patient prognosis, simultaneously. © Springer Japan 2012.


Kurogochi M.,Hokkaido University | Matsushista T.,Hokkaido University | Amano M.,Hokkaido University | Furukawa J.-I.,Hokkaido University | And 4 more authors.
Molecular and Cellular Proteomics | Year: 2010

Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. However, in-depth knowledge of protein glycosylation to uncover functions and their clinical applications requires quantitative glycoproteomics eliciting both peptide and glycan sequences concurrently. Here we describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). LC-MS/MS analysis of de-glycosylated peptides identified 270 mouse serum peptides (95 glycoproteins) as sialylated glycopeptides, of which 67 glycopeptides were fully characterized by MS/MS analyses in a straightforward manner. We revealed the importance of a fragment ion containing innermost N-acetylglucosamine (GlcNAc) residue as MRM transitions regardless the sequence of the peptides. Versatility of the reverse glycoblotting-assisted MRM assays was demonstrated by quantitative comparison of 25 targeted glycopeptides from 16 proteins between mice with homo and hetero types of diabetes disease model. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Hirose K.,Hokkaido University | Amano M.,Hokkaido University | Hashimoto R.,Hokkaido University | Lee Y.C.,Johns Hopkins University | And 3 more authors.
Biochemistry | Year: 2011

A large set of glycome information was obtainedfrom egg white proteins of 88 samples from Galloanserae (63 Anseriformes and 25 Galliformes). The data were obtained on whole N-glycan structures and types of sialic acids of these egg whites by glycoblotting-based high-throughput and quantitative glycomics. The results revealed clear trends and complexity patterns as well as diversity among taxonomic groups. It is wellknown that chicken, a representative domesticated poultry involved in Galliformes, can become an influenza host.However, our data demonstrate that duck, wild goose, and swan of Anseriformes are representative migratory birds that are known as natural hosts of the influenza virus. Hierarchical clustering analysis of the expression pattern of N-glycome (total of 61 N-glycan peaks) revealed that the members of Galloanserae can be classified into two major groups and five submajor clusters (clusters 1-5) on the basis of simple m/z values obtained by MALDITOF MS. It is clear that expression patterns of N-glycomes in the five clusters are influenced significantly by the features such as the body size of the birds, rather than by the difference of the family. On the other hand, quantitative analysis showed that the total amounts of sialic acids in egg whites of Galliformes were distinctly larger than those of Anseriformes. However, it was also revealed in Anseriformes that Neu5Gc and KDN, in addition to common Neu5Ac, were expressed significantly in both N- and O-glycans of glycoproteins and glycosphingolipids, suggesting the influence of their lifestyles and diet. This is the first report that KDN exists in egg white. These results and the environmental factors are discussed preliminarily with respect to their evolutionary lineage. © 2011 American Chemical Society.


Valadez-Vega C.,Autonomous University of the State of Hidalgo | Alvarez-Manilla G.,Ezose Sciences | Riveron-Negrete L.,Parasitologia Experimental | Garcia-Carranca A.,National Autonomous University of Mexico | And 8 more authors.
Molecules | Year: 2011

Lectins comprise a heterogeneous class of proteins that recognize the carbohydrate moieties of glycoconjugates with high specificity. Numerous studies have shown that lectins are capable of recognizing specific carbohydrate moieties displayed by malignant cells or tissues. The present work was performed to investigate the effects of tepary bean (Phaseolus acutifolius) lectins on proliferation, colony formation, and alteration of DNA synthesis of human malignant cells. Tepary bean lectin showed dose dependent effects on the inhibition of viability as well as on colony formation in two human malignant cells lines (C33-A, Sw480); By contrast, tepary bean lectin only showed significant effects on DNA synthesis on Sw480 cells. Our results provide evidence of the anti- proliferative and cytotoxic effects of the tepary bean lectins on C33-A and Sw480 cells lines. © 2011.


Miura Y.,Ezose Sciences | Kato K.,Hokkaido University | Takegawa Y.,Hokkaido University | Kurogochi M.,Hokkaido University | And 7 more authors.
Analytical Chemistry | Year: 2010

Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hydrazide-polymers/ solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing O-glycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers. © 2010 American Chemical Society.


Amano M.,Hokkaido University | Yamaguchi M.,Hokkaido University | Takegawa Y.,Hokkaido University | Yamashita T.,Hokkaido University | And 9 more authors.
Molecular and Cellular Proteomics | Year: 2010

Although various glycoforms appear to participate independently in multiple molecular interactions in cellular adhesion that contribute to embryogenesis and organogenesis, a full portrait of the glycome diversity and the effect of the structural variations of cellular glycoforms on individual cell stages in proliferation and differentiation remain unclear. Here we describe a novel concept for the characterization of dynamic glycoform alteration during cell differentiation by means of "glycoblotting-based cellular glycomics," the only method allowing for rapid and quantitative glycan analysis. We demonstrated that processes of dynamic cellular differentiation of mouse embryonic carcinoma cells, P19CL6 and P19C6, and mouse embryonic stem cells into cardiomyocytes or neural cells can be monitored and characterized quantitatively by profiling entire N-glycan structures of total cell glycoproteins. Whole N-glycans enriched and identified by the glycoblotting method (67 glycans for P19CL6, 75 glycans for P19C6, and 72 glycans for embryonic stem cells) were profiled and bar-coded quantitatively with respect to the ratio of subgroups composed of characteristic glycoforms, namely glycotypes. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Nakahara T.,Ezose Sciences
Trends in Glycoscience and Glycotechnology | Year: 2013

Research in glycomics and glycoproteomics is making rapid progress as a result of technological advances in mass spectrometry. Bioinformatics is an indispensable discipline for analyzing overwhelming amounts of information generated by mass spectrometry. This minireview summarizes bioinformatics contributions to glycan mass spectrometry data analysis. To simplify the discussion on the wide range of bioinformatics contributions to this field, the research shown here will be categorized into three areas; "Calculator-type tools," "Technologies for glycan structure determination by tandem MS" and "Other noteworthy bioinformatics technologies for MS." Lastly, future prospects of the contribution of bioinformatics to glycobiology will be discussed. © 2013 FCCA (Forum: Carbohydrates Coming of Age).


Cancer-associated O-glycopeptide combination epitopes derived from the VNTR of MUC1 are disclosed. Autoantibodies present in human sera target the combination epitopes and are reduced or absent in cancer patients. The epitopes are useful as therapeutic and immunoprophylactic cancer vaccines. Monoclonal antibodies directed against the epitopes are also useful as immunotherapeutics for treatment and prevention of cancer. Diagnostic methods using the epitopes and antibodies are also disclosed.


The objectives of this study were to examine the whole-serum N-glycan profile of patients with unresectable pancreatic cancer and to evaluate the ability of glycans to predict gemcitabine treatment efficacy and patient survival.We collected serum from 52 patients with advanced pancreatic cancer before they began gemcitabine monotherapy. The serum glycan profile was measured through comprehensive quantitative high-throughput glycome analysis and compared with the treatment efficacy and patient survival.Of the 61 glycans detected, the serum levels of glycan 4310 (molecular weight [m/z] 1549.566), 6301 (m/z 2032.724), and 9200 (m/z 2010.692) were high in patients with a short time to tumor progression (TTP). Multivariate analysis revealed that a high glycan 9200 concentration was an independent risk factor for shorter TTP (hazard ratio, 2.11; 95% confidence interval, 1.07-4.17) and poor overall survival (hazard ratio, 2.56; 95% confidence interval, 1.08-6.19). The median TTP of patients with up-regulation of 9200 after gemcitabine treatment was shorter than for the remaining patients (91 vs 301 days; P = 0.0005). A similar relationship was observed for overall survival (median, 181 vs 561 days; P = 0.001).Glycan 9200 is a possible biomarker predicting gemcitabine efficacy survival in patients with unresectable pancreatic cancer.


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