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1.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800786

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is known as a highly aggressive malignant disease. Prognosis for patients is notoriously poor, despite improvements in surgical techniques and new (neo)adjuvant chemotherapy regimens. Early detection of PDAC may increase the overall survival. It is furthermore foreseen that precision medicine will provide improved prognostic stratification and prediction of therapeutic response. In this review, omics-based discovery efforts are presented that aim for novel diagnostic and prognostic biomarkers of PDAC. For this purpose, we systematically evaluated the literature published between 1999 and 2020 with a focus on protein- and protein-glycosylation biomarkers in pancreatic cancer patients. Besides genomic and transcriptomic approaches, mass spectrometry (MS)-based proteomics and glycomics of blood- and tissue-derived samples from PDAC patients have yielded new candidates with biomarker potential. However, for reasons discussed in this review, the validation and clinical translation of these candidate markers has not been successful. Consequently, there has been a change of mindset from initial efforts to identify new unimarkers into the current hypothesis that a combination of biomarkers better suits a diagnostic or prognostic panel. With continuing development of current research methods and available techniques combined with careful study designs, new biomarkers could contribute to improved detection, prognosis, and prediction of pancreatic cancer.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma Ductal Pancreático/diagnóstico , Glicômica/métodos , Glicoproteínas/análise , Espectrometria de Massas/métodos , Proteínas de Neoplasias/análise , Neoplasias Pancreáticas/diagnóstico , Proteômica/métodos , Líquidos Corporais/química , Carcinoma Ductal Pancreático/química , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Tomada de Decisão Clínica , Diagnóstico Diferencial , Detecção Precoce de Câncer , Humanos , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/epidemiologia , Síndromes Neoplásicas Hereditárias/genética , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Pancreatite Crônica/diagnóstico , Lesões Pré-Cancerosas/diagnóstico , Medicina de Precisão , Valor Preditivo dos Testes , Prognóstico , Reprodutibilidade dos Testes , Fatores de Risco
3.
Nat Commun ; 11(1): 5153, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33056991

RESUMO

Correlation networks are frequently used to statistically extract biological interactions between omics markers. Network edge selection is typically based on the statistical significance of the correlation coefficients. This procedure, however, is not guaranteed to capture biological mechanisms. We here propose an alternative approach for network reconstruction: a cutoff selection algorithm that maximizes the overlap of the inferred network with available prior knowledge. We first evaluate the approach on IgG glycomics data, for which the biochemical pathway is known and well-characterized. Importantly, even in the case of incomplete or incorrect prior knowledge, the optimal network is close to the true optimum. We then demonstrate the generalizability of the approach with applications to untargeted metabolomics and transcriptomics data. For the transcriptomics case, we demonstrate that the optimized network is superior to statistical networks in systematically retrieving interactions that were not included in the biological reference used for optimization.


Assuntos
Algoritmos , Glicômica/métodos , Metabolômica/métodos , RNA-Seq/métodos , Interpretação Estatística de Dados , Glicômica/estatística & dados numéricos , Humanos , Imunoglobulina G/metabolismo , Metabolômica/estatística & dados numéricos , RNA-Seq/estatística & dados numéricos
4.
J Am Soc Mass Spectrom ; 31(10): 2013-2024, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-32880453

RESUMO

As corona virus disease 2019 (COVID-19) is a rapidly growing public health crisis across the world, our knowledge of meaningful diagnostic tests and treatment for severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) is still evolving. This novel coronavirus disease COVID-19 can be diagnosed using RT-PCR, but inadequate access to reagents, equipment, and a nonspecific target has slowed disease detection and management. Precision medicine, individualized patient care, requires suitable diagnostics approaches to tackle the challenging aspects of viral outbreaks where many tests are needed in a rapid and deployable approach. Mass spectrometry (MS)-based technologies such as proteomics, glycomics, lipidomics, and metabolomics have been applied in disease outbreaks for identification of infectious disease agents such as virus and bacteria and the molecular phenomena associated with pathogenesis. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is widely used in clinical diagnostics in the United States and Europe for bacterial pathogen identification. Paper spray ionization mass spectrometry (PSI-MS), a rapid ambient MS technique, has recently open a new opportunity for future clinical investigation to diagnose pathogens. Ultra-high-pressure liquid chromatography coupled high-resolution mass spectrometry (UHPLC-HRMS)-based metabolomics and lipidomics have been employed in large-scale biomedical research to discriminate infectious pathogens and uncover biomarkers associated with pathogenesis. PCR-MS has emerged as a new technology with the capability to directly identify known pathogens from the clinical specimens and the potential to identify genetic evidence of undiscovered pathogens. Moreover, miniaturized MS offers possible applications with relatively fast, highly sensitive, and potentially portable ways to analyze for viral compounds. However, beneficial aspects of these rapidly growing MS technologies in pandemics like COVID-19 outbreaks has been limited. Hence, this perspective gives a brief of the existing knowledge, current challenges, and opportunities for MS-based techniques as a promising avenue in studying emerging pathogen outbreaks such as COVID-19.


Assuntos
Infecções por Coronavirus/etiologia , Lipidômica/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Pneumonia Viral/etiologia , Proteômica/métodos , Cromatografia Líquida de Alta Pressão , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Glicômica/métodos , Humanos , Pandemias , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
Nat Protoc ; 15(8): 2668-2704, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32681150

RESUMO

The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocalyx is thus essential to understanding cell physiology and elucidating its role in promoting health and disease. This protocol describes how to comprehensively characterize the glycocalyx N-glycans and O-glycans of glycoproteins, as well as intact glycolipids in parallel, using the same enriched membrane fraction. Profiling of the glycans and the glycolipids is performed using nanoflow liquid chromatography-mass spectrometry (nanoLC-MS). Sample preparation, quantitative LC-tandem MS (LC-MS/MS) analysis, and data processing methods are provided. In addition, we discuss glycoproteomic analysis that yields the site-specific glycosylation of membrane proteins. To reduce the amount of sample needed, N-glycan, O-glycan, and glycolipid analyses are performed on the same enriched fraction, whereas glycoproteomic analysis is performed on a separate enriched fraction. The sample preparation process takes 2-3 d, whereas the time spent on instrumental and data analyses could vary from 1 to 5 d for different sample sizes. This workflow is applicable to both cell and tissue samples. Systematic changes in the glycocalyx associated with specific glycoforms and glycoconjugates can be monitored with quantitation using this protocol. The ability to quantitate individual glycoforms and glycoconjugates will find utility in a broad range of fundamental and applied clinical studies, including glycan-based biomarker discovery and therapeutics.


Assuntos
Glicocálix/metabolismo , Glicômica/métodos , Linhagem Celular , Humanos , Polissacarídeos/metabolismo
7.
PLoS One ; 15(2): e0228507, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045434

RESUMO

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is essential for the maintenance of pregnancy. Glycosylation of hCG is known to be essential for its biological activity. "Hyperglycosylated" variants secreted during early pregnancy have been proposed to be involved in initial implantation of the embryo and as a potential diagnostic marker for gestational diseases. However, what constitutes "hyperglycosylation" is not yet fully understood. In this study, we perform comparative N-glycomic analysis of hCG expressed in the same individuals during early and late pregnancy to help provide new insights into hCG function, reveal new targets for diagnostics and clarify the identity of hyperglycosylated hCG. hCG was isolated in urine collected from women at 7 weeks and 20 weeks' gestation. hCG was also isolated in urine from women diagnosed with gestational trophoblastic disease (GTD). We used glycomics methodologies including matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS methods to characterise the N-glycans associated with hCG purified from the individual samples. The structures identified on the early pregnancy (EP-hCG) and late pregnancy (LP-hCG) samples corresponded to mono-, bi-, tri-, and tetra-antennary N-glycans. A novel finding was the presence of substantial amounts of bisected type N-glycans in pregnancy hCG samples, which were present at much lower levels in GTD samples. A second novel observation was the presence of abundant LewisX antigens on the bisected N-glycans. GTD-hCG had fewer glycoforms which constituted a subset of those found in normal pregnancy. When compared to EP-hCG, GTD-hCG samples had decreased signals for tri- and tetra-antennary N-glycans. In terms of terminal epitopes, GTD-hCG had increased signals for sialylated structures, while LewisX antigens were of very minor abundance. hCG carries the same N-glycans throughout pregnancy but in different proportions. The N-glycan repertoire is more diverse than previously reported. Bisected and LewisX structures are potential targets for diagnostics. hCG isolated from pregnancy urine inhibits NK cell cytotoxicity in vitro at nanomolar levels and bisected type glycans have previously been implicated in the suppression of NK cell cytotoxicity, suggesting that hCG-related bisected type N-glycans may directly suppress NK cell cytotoxicity.


Assuntos
Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Carboidratos , Gonadotropina Coriônica Humana Subunidade beta/sangue , Gonadotropina Coriônica Humana Subunidade beta/urina , Feminino , Idade Gestacional , Doença Trofoblástica Gestacional/sangue , Doença Trofoblástica Gestacional/metabolismo , Doença Trofoblástica Gestacional/urina , Glicômica/métodos , Glicosilação , Humanos , Gravidez , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
8.
J Chromatogr A ; 1619: 460934, 2020 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-32029268

RESUMO

Peptide-N-glycosidase F (PNGase F) is the most frequently used enzyme to release N-glycan from glycoproteins in glycomics; however, the releasing process using PNGase F is tedious and can range in duration from hours to overnight. Recently, efforts have been made to accelerate this enzymatic reaction, and they include the use of microwave irradiation, ultrahigh pressure, enzyme immobilization, and other techniques. Here, we developed a novel method combining the oriented immobilization of PNGase F on magnetic particles and microwave-assisted enzymatic digestion techniques to achieve highly efficient release of N-glycans. The oriented immobilization of PNGase F on magnetic particles utilizes the affinity of its co-expressed His-tag towards iminodiacetic acid-Nickel modified magnetic particles. Compared with non-oriented immobilization, the oriented immobilization of PNGase F exhibits several advantages including tolerance to high temperature (52 °C) and the ability to retain strong activity after more than five reuses. When used in combination with microwave irradiation, efficient N-glycan removal from ribonuclease B was achieved within 5 min. The proposed strategy was also used to release glycan from fetuin and human serum and has proven to provide a promising deglycosylation method for the characterization of protein glycosylation.


Assuntos
Glicômica/métodos , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Polissacarídeos/metabolismo , Enzimas Imobilizadas/metabolismo , Fetuínas/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/química , Micro-Ondas , Polissacarídeos/efeitos da radiação , Ribonucleases/metabolismo
9.
J Biol Chem ; 295(10): 3173-3188, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32001617

RESUMO

Echinoderms are among the most primitive deuterostomes and have been used as model organisms to understand chordate biology because of their close evolutionary relationship to this phylogenetic group. However, there are almost no data available regarding the N-glycomic capacity of echinoderms, which are otherwise known to produce a diverse set of species-specific glycoconjugates, including ones heavily modified by fucose, sulfate, and sialic acid residues. To increase the knowledge of diversity of carbohydrate structures within this phylum, here we conducted an in-depth analysis of N-glycans from a brittle star (Ophiactis savignyi) as an example member of the class Ophiuroidea. To this end, we performed a multi-step N-glycan analysis by HPLC and various exoglyosidase and chemical treatments in combination with MALDI-TOF MS and MS/MS. Using this approach, we found a wealth of hybrid and complex oligosaccharide structures reminiscent of those in higher vertebrates as well as some classical invertebrate glycan structures. 70% of these N-glycans were anionic, carrying either sialic acid, sulfate, or phosphate residues. In terms of glycophylogeny, our data position the brittle star between invertebrates and vertebrates and confirm the high diversity of N-glycosylation in lower organisms.


Assuntos
Glicômica/métodos , Polissacarídeos/química , Estrelas-do-Mar/metabolismo , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Glicosídeo Hidrolases/metabolismo , Glicosilação , Oligossacarídeos/química , Filogenia , Polissacarídeos/classificação , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estrelas-do-Mar/classificação
10.
J Vis Exp ; (155)2020 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-32009638

RESUMO

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease susceptibility, drug target discovery, and precision medicine. Alternative IgG N-glycans have been reported in several common chronic diseases and suggested to have great potential in clinical applications (i.e., biomarkers for diagnosis and prediction of diseases). IgG N-glycans are widely characterized using the method of hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography (UPLC). UPLC is a stable detection technology with good reproducibility and high relative quantitative accuracy. In addition, the structure of IgG N-glycan is clearly separated, and glycan composition and relative abundance in plasma are characterized.


Assuntos
Cromatografia Líquida/métodos , Glicômica/métodos , Imunoglobulina G/sangue , Humanos
11.
Sci Rep ; 10(1): 671, 2020 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959827

RESUMO

A common renal disease, immunoglobulin A (IgA) nephropathy (IgAN), is associated with glomerular deposition of IgA1-containing immune complexes. IgA1 hinge region (HR) has up to six clustered O-glycans consisting of Ser/Thr-linked N-acetylgalactosamine with ß1,3-linked galactose and variable sialylation. IgA1 glycoforms with some galactose-deficient (Gd) HR O-glycans play a key role in IgAN pathogenesis. The clustered and variable O-glycans make the IgA1 glycomic analysis challenging and better approaches are needed. Here, we report a comprehensive analytical workflow for IgA1 HR O-glycoform analysis. We combined an automated quantitative analysis of the HR O-glycopeptide profiles with sequential deglycosylation to remove all but Gd O-glycans from the HR. The workflow was tested using serum IgA1 from healthy subjects. Twelve variants of glycopeptides corresponding to the HR with three to six O-glycans were detected; nine glycopeptides carried up to three Gd O-glycans. Sites with Gd O-glycans were unambiguously identified by electron-transfer/higher-energy collision dissociation tandem mass spectrometry. Extracted ion chromatograms of isomeric glycoforms enabled quantitative assignment of Gd sites. The most frequent Gd site was T236, followed by S230, T233, T228, and S232. The new workflow for quantitative profiling of IgA1 HR O-glycoforms with site-specific resolution will enable identification of pathogenic IgA1 HR O-glycoforms in IgAN.


Assuntos
Glomerulonefrite por IGA/etiologia , Glicômica/métodos , Glicopeptídeos/química , Ensaios de Triagem em Larga Escala/métodos , Imunoglobulina A/química , Imunoglobulina A/metabolismo , Polissacarídeos/química , Glicosilação , Humanos , Isoformas de Proteínas , Espectrometria de Massas em Tandem
13.
Mol Cell Proteomics ; 19(1): 11-30, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31591262

RESUMO

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.


Assuntos
Anticorpos Monoclonais/química , Produtos Biológicos , Biofarmácia/métodos , Anticorpos Monoclonais/metabolismo , Glicômica/métodos , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Laboratórios , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos
14.
J Pharm Biomed Anal ; 178: 112892, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31593866

RESUMO

As glycomics research is gaining momentum in the biopharmaceutical industry, there is an increasing need for reproducible high throughput glycoanalytical methods to monitor and characterize the N-glycosylation of therapeutic glycoproteins. Since the glycosylation pattern of glycobiotherapeutics influences their important biological functions, approaches to comprehensively analyze these complex molecules is of high importance. This paper reports on the use of multicapillary gel electrophoresis in high throughput analysis of fluorophore labeled partitioned N-glycan libraries to generate a new glucose unit database that was consequently applied to identify the carbohydrate structures of two high profile biopharmaceuticals, adalimumab and etanercept.


Assuntos
Adalimumab/química , Eletroforese Capilar/métodos , Etanercepte/química , Glucose/química , Bases de Dados Factuais , Glicômica/métodos , Glicoproteínas/química , Glicosilação , Ensaios de Triagem em Larga Escala , Reprodutibilidade dos Testes
16.
Integr Biol (Camb) ; 11(7): 315-329, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31712825

RESUMO

Commensal bacteria must colonize host mucosal surfaces to exert health-promoting properties, and bind to gastrointestinal tract (GIT) mucins via their cell surface adhesins. Considerable effort has been directed towards discovery of pathogen adhesins and their ligands to develop anti-infective strategies; however, little is known about the lectin-like adhesins and associated carbohydrate ligands in commensals. In this study, an in silico approach was used to detect surface exposed adhesins in the human commensal Lactobacillus paracasei subsp. paracasei, a promising probiotic commonly used in dairy product fermentation that presents anti-microbial activity. Of the 13 adhesin candidates, 3 sortase-dependent pili clusters were identified in this strain and expression of the adhesin candidate genes was confirmed in vitro. Mass spectrometry analysis confirmed the presence of surface adhesin elongation factor Tu and the chaperonin GroEL, but not pili expression. Whole cells were subsequently incubated on microarrays featuring a panel of GIT mucins from nine different mammalian species and two human-derived cell lines and a library of carbohydrate structures. Binding profiles were compared to those of two known pili-producing lactobacilli, L. johnsonii and L. rhamnosus and all Lactobacillus species displayed overlapping but distinct signatures, which may indicate different abilities for regiospecific GIT colonization. In addition, L. paracasei whole cells favoured binding to α-(2 â†’ 3)-linked sialic acid and α-(1 â†’ 2)-linked fucose-containing carbohydrate structures including blood groups A, B and O and Lewis antigens x, y and b. This study furthers our understanding of host-commensal cross-talk by identifying potential adhesins and specific GIT mucin and carbohydrate ligands and provides insight into the selection of colonization sites by commensals in the GIT.


Assuntos
Adesinas Bacterianas/química , Carboidratos/química , Microbioma Gastrointestinal , Glicômica/métodos , Lactobacillus paracasei , Animais , Aderência Bacteriana , Simulação por Computador , Fucose/química , Humanos , Lactobacillus , Lactobacillus johnsonii , Lactobacillus rhamnosus , Ligantes , Técnicas de Sonda Molecular , Probióticos , Ligação Proteica , RNA Bacteriano/isolamento & purificação , Propriedades de Superfície
17.
Sci Rep ; 9(1): 17361, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31758065

RESUMO

Breast cancer brain metastasis has been recognized as one of the central issues in breast cancer research. The elucidation of the processes and pathways that mediate this step will provide important clues for a better understanding of breast cancer metastasis. Increasing evidence suggests that aberrant glycosylation patterns greatly contribute to cell invasion and cancer metastasis. Herein, we combined next-generation RNA sequencing with liquid chromatography-tandem mass spectrometry-based proteomic and N-glycomic analysis from five breast cancer cell lines and one brain cancer cell line to investigate the possible mechanisms of breast cancer brain metastasis. The genes/proteins associated with cell movement were highlighted in breast cancer brain metastasis. The integrin signaling pathway and the up-regulation of α-integrin (ITGA2, ITGA3) were associated with the brain metastatic process. 12 glycogenes showed unique expression in 231BR, which could result in an increase of sialylation during brain metastasis. In agreement with the changes of glycogenes, 60 out of 63 N-glycans that were identified exhibited differential expression among cell lines. The correlation between glycogenes and glycans revealed the importance of sialylation and sialylated glycans in breast cancer brain metastasis. Highly sialylated N-glycans, which were up-regulated in brain-seeking cell line 231BR, likely play a role in brain metastasis.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Integrinas/metabolismo , Polissacarídeos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Cromatografia Líquida , Feminino , Perfilação da Expressão Gênica/métodos , Glicômica/métodos , Glicosilação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Integrinas/análise , Polissacarídeos/análise , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Integração de Sistemas , Espectrometria de Massas em Tandem , Transcriptoma/fisiologia , Regulação para Cima
18.
Anal Chim Acta ; 1091: 1-22, 2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31679562

RESUMO

N-glycosylation is one of the most frequently occurring protein post-translational modifications (PTMs) with broad cellular, physiological and pathological relevance. Mass spectrometry-based N-glycomics has become the state-of-the-art instrumental analytical pipeline for sensitive, high-throughput and comprehensive characterization of N-glycans and N-glycomes. Improvement and new development of methods in N-glycan release, enrichment, derivatization, isotopic labeling, separation, ionization, MS, tandem MS and informatics accompany side-by-side wider and deeper application. This review provides a comprehensive update of mass spectrometry-based qualitative and quantitative N-glycomics in the years of 2017-2018.


Assuntos
Glicômica/métodos , Polissacarídeos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Sequência de Carboidratos , Linhagem Celular , Humanos , Vírus da Influenza A Subtipo H9N2/química
19.
Bioconjug Chem ; 30(11): 2897-2908, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31600064

RESUMO

To aid in generating complex and diverse natural glycan libraries for functional glycomics, more efficient and reliable methods are needed to derivatize glycans. Here we present our development of a reversible, cleavable bifunctional linker 3-(methoxyamino)propylamine (MAPA). As the fluorenylmethyloxycarbonate (Fmoc) version (F-MAPA), it is highly fluorescent and efficiently derivatizes free reducing glycans to generate closed-ring derivatives that preserve the structural integrity of glycans. A library of glycans were derivatized and used to generate a covalent glycan microarray using N-hydroxysuccinimide derivatization. The array was successfully interrogated by a variety of lectins and antibodies, demonstrating the importance of closed-ring chemistry. The glycan derivatization was also performed at large scale using milligram quantities of glycans and excess F-MAPA, and the reaction system was successfully recycled up to five times, without an apparent decrease in conjugation efficiency. The MAPA-glycan is also easy to link to protein to generate neoglycoproteins with equivalent glycan densities. Importantly, the MAPA linker can be reversibly cleaved to regenerate free reducing glycans for detailed structural analysis (catch-and-release), often critical for functional studies of undefined glycans from natural sources. The high conjugation efficiency, bright fluorescence, and reversible cleavage of the linker enable access to natural glycans for functional glycomics.


Assuntos
Fluorescência , Glicômica/métodos , Glicoproteínas/química , Polissacarídeos/química , Propilaminas/química , Configuração de Carboidratos , Humanos , Análise em Microsséries
20.
J Proteome Res ; 18(11): 3985-3998, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31566983

RESUMO

Lung cancer is the leading cause of cancer death in women living in the United States, which accounts for approximately the same percentage of cancer deaths in women as breast, ovary, and uterine cancers combined. Targeted blood plasma glycomics represents a promising source of noninvasive diagnostic and prognostic biomarkers for lung cancer. Here, 208 samples from lung cancer patients and 207 age-matched controls enrolled in the Women Epidemiology Lung Cancer (WELCA) study were analyzed by a bottom-up glycan "node" analysis approach. Glycan features, quantified as single analytical signals, including 2-linked mannose, α2-6 sialylation, ß1-4 branching, ß1-6 branching, 4-linked GlcNAc, and antennary fucosylation, exhibited abilities to distinguish cases from controls (ROC AUCs: 0.68-0.92) and predict survival in patients (hazard ratios: 1.99-2.75) at all stages. Notable alterations of glycan features were observed in stages I-II. Diagnostic and prognostic glycan features were mostly independent of smoking status, age, gender, and histological subtypes of lung cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Glicômica/métodos , Neoplasias Pulmonares/metabolismo , Polissacarídeos/metabolismo , Idoso , Biomarcadores Tumorais/sangue , Estudos de Casos e Controles , Feminino , Glicosilação , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/epidemiologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Polissacarídeos/sangue , Prognóstico , Curva ROC , Análise de Sobrevida
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