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1.
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
2.
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
3.
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
4.
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
5.
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
6.
PLoS One ; 14(10): e0223270, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31589631

RESUMO

The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated.


Assuntos
Glicômica/métodos , Glicoproteínas/química , Polissacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Glicômica/normas , Células HCT116 , Células HT29 , Humanos , Imunoglobulina G/química , Polissacarídeos/química , Espectrometria de Massas por Ionização por Electrospray/normas
7.
Anal Chim Acta ; 1089: 90-99, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31627822

RESUMO

N-glycans are involved in a variety of biological processes and associated with many diseases. However, the sensitive and specific detection of glycans via mass spectrometry (MS) remains challenging due to their relatively low abundance and low ionization efficiency. In this work, a new method termed glycan reductive amino acid coded affinity tagging (GRACAT) is developed to address these problems. Through labeling the reducing ends of glycan with a biotinylated arginine, the ionization efficiency of glycan was increased nearly 50-fold and glycan isomers were discriminated by improved signals of fragments in tandem spectra. Moreover, the strong affinity interaction between streptavidin and biotin enabled highly specific enrichment of the biotin-tagged glycans from the mixture of proteins and peptides. We successfully applied GRACAT in the profiling of N-glycome in human serum and bovine milk, in which a total of 55 and 67 N-glycans were detected respectively.


Assuntos
Marcadores de Afinidade/química , Glicômica/métodos , Leite/química , Polissacarídeos/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Biotina/análogos & derivados , Bovinos , Dipeptídeos/química , Humanos , Extração em Fase Sólida , Estreptavidina/química
8.
Anal Chim Acta ; 1081: 112-119, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446948

RESUMO

Accurate, simple and economical methods for quantifying N-glycans are continuously required for discovering disease biomarkers and quality control of biopharmaceuticals. Quantitative N-glycomics based on MS using exogenous isotopic labeling internal standards is promising as it is simple and accurate. However, it is largely hampered by the lack of available glycan internal standard libraries with good coverage of the natural glycan structural heterogeneity as well as broad dynamic mass and ion abundance range. To overcome this limitation, we developed a novel method, providing 'Bionic Glycome' as internal standards for glycan quantitation by MALDI-MS. Bionic Glycome was produced using N-glycome from pooled samples to be analyzed as substrate by one step of glycan reducing and isotope labeling (Glycan-RAIL). Each bionic glycan has 3 Da mass increment over its corresponding glycan analyte based on hemiacetals/alditols and H/D mass difference. In addition, Bionic Glycome has the same glycome composition and similar glycome profile in abundance with N-glycome to be analyzed from biological sample. Through the investigation of single glycan standard and complex glycans released from model glycoprotein and serum, the results demonstrate that the method has good quantitative accuracy and high reproducibility. Lastly, this method was successfully used for discovery of lung cancer specific glycan markers by comparing the serum glycans from each sample in lung cancer group (n = 16) and healthy controls (n = 16), indicating its potential in clinical applications.


Assuntos
Biomarcadores Tumorais/análise , Biomarcadores Tumorais/normas , Polissacarídeos/análise , Polissacarídeos/normas , Idoso , Biomarcadores Tumorais/química , Boroidretos/química , Deutério , Feminino , Glicômica/métodos , Humanos , Marcação por Isótopo , Neoplasias Pulmonares/química , Masculino , Pessoa de Meia-Idade , Oxirredução , Polissacarídeos/química , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
9.
J Agric Food Chem ; 67(37): 10543-10551, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31464438

RESUMO

Adulteration of meat and meat products causes concerns to consumers. It is necessary to develop novel robust and sensitive methods that can authenticate the origin of meat by qualitative and quantitative means to minimize the drawbacks of the existing methods. This study has shown that the protein N-glycosylation profiles of different meats are species specific and thus can be used for meat authentication. Based on the N-glycan pattern, the investigated five meat species (beef, chicken, pork, duck, and mutton) can be distinguished by principal component analysis, and partial least square regression was performed to build a calibration and validation model for the prediction of adulteration ratio. Using this method, beef samples adulterated with a lower-value duck meat could be detected down to the addition ratio as low as 2.2%. The most distinguishing N-glycans from beef and duck were elucidated for the detailed structures.


Assuntos
Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Glicômica/métodos , Polissacarídeos/química , Animais , Bovinos , Galinhas , Análise Discriminante , Patos , Glicosilação , Carne , Análise de Componente Principal , Carne Vermelha , Suínos
10.
Talanta ; 205: 120104, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450448

RESUMO

Body fluid N-glycome analysis as well as glyco-proteoform profiling of existing protein biomarkers potentially provides a stratification layer additional to quantitative, diagnostic protein levels. For clinical omics applications, the collection of a dried blood spot (DBS) is increasingly pursued as an alternative to sampling milliliters of peripheral blood. Here we evaluate DBS cards as a blood collection strategy for protein N-glycosylation analysis aiming for high-throughput clinical applications. A protocol for facile N-glycosylation profiling from DBS is developed that includes sialic acid linkage differentiation. This protocol is based on a previously established total plasma N-glycome mass spectrometry (MS) method, with adjustments for the analysis of DBS specimens. After DBS-punching and protein solubilization N-glycans are released, followed by chemical derivatization of sialic acids and MS-measurement of N-glycan profiles. With this method, more than 80 different glycan structures are identified from a DBS, with RSDs below 10% for the ten most abundant glycans. N-glycan profiles of finger-tip blood and venous blood are compared and short-term stability of DBS is demonstrated. This method for fast N-glycosylation profiling of DBS provides a minimally invasive alternative to conventional serum and plasma protein N-glycosylation workflows. With simplified blood sampling this DBS approach has vast potential for clinical glycomics applications.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Glicômica/métodos , Polissacarídeos/sangue , Humanos , Polissacarídeos/química , Ácidos Siálicos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
11.
Molecules ; 24(16)2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31443278

RESUMO

For the effective discovery of the biological roles and disease-specific alterations concerning protein glycosylation in tissue samples, it is important to know beforehand the quantitative and qualitative variations of glycan structures expressed in various types of cells, sites, and tissues. To this end, we used laser microdissection-assisted lectin microarray (LMA) to establish a simple and reproducible method for high-throughput and in-depth glycomic profiling of formalin-fixed paraffin-embedded tissue sections. Using this "tissue glycome mapping" approach, we present 234 glycomic profiling data obtained from nine tissue sections (pancreas, heart, lung, thymus, gallbladder, stomach, small intestine, colon, and skin) of two 8-week-old male C57BL/6J mice. We provided this LMA-based dataset in the similar interface as that of GlycomeAtlas, a previously developed tool for mass spectrometry-based tissue glycomic profiling, allowing easy comparison of the two types of data. This online tool, called "LM-GlycomeAtlas", allows users to visualize the LMA-based tissue glycomic profiling data associated with the sample information as an atlas. Since the present dataset allows the comparison of glycomic profiles, it will facilitate the evaluation of site- and tissue-specific glycosylation patterns. Taking advantage of its extensibility, this tool will continue to be updated with the expansion of deposited data.


Assuntos
Glicômica , Lectinas/metabolismo , Análise Serial de Proteínas , Software , Interface Usuário-Computador , Animais , Glicômica/métodos , Glicosilação , Masculino , Camundongos , Microdissecção , Especificidade de Órgãos , Análise Serial de Proteínas/métodos
12.
Nat Commun ; 10(1): 3275, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332201

RESUMO

The mass spectrometry (MS)-based analysis of free polysaccharides and glycans released from proteins, lipids and proteoglycans increasingly relies on databases and software. Here, we review progress in the bioinformatics analysis of protein-released N- and O-linked glycans (N- and O-glycomics) and propose an e-infrastructure to overcome current deficits in data and experimental transparency. This workflow enables the standardized submission of MS-based glycomics information into the public repository UniCarb-DR. It implements the MIRAGE (Minimum Requirement for A Glycomics Experiment) reporting guidelines, storage of unprocessed MS data in the GlycoPOST repository and glycan structure registration using the GlyTouCan registry, thereby supporting the development and extension of a glycan structure knowledgebase.


Assuntos
Biologia Computacional/métodos , Glicômica/métodos , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Animais , Biologia Computacional/normas , Bases de Dados Factuais/normas , Bases de Dados Factuais/estatística & dados numéricos , Humanos , Espectrometria de Massas/métodos , Padrões de Referência
13.
Glycoconj J ; 36(4): 241-257, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31267247

RESUMO

We have explored the fundamental biological processes by which complex carbohydrates expressed on cellular glycoproteins and glycolipids and in secretions of cells promote cell adhesion and signaling. We have also explored processes by which animal pathogens, such as viruses, bacteria, and parasites adhere to glycans of animal cells and initiate disease. Glycans important in cell signaling and adhesion, such as key O-glycans, are essential for proper animal development and cellular differentiation, but they are also involved in many pathogenic processes, including inflammation, tumorigenesis and metastasis, and microbial and parasitic pathogenesis. The overall hypothesis guiding these studies is that glycoconjugates are recognized and bound by a growing class of proteins called glycan-binding proteins (GBPs or lectins) expressed by all types of cells. There is an incredible variety and diversity of GBPs in animal cells involved in binding N- and O-glycans, glycosphingolipids, and proteoglycan/glycosaminoglycans. We have specifically studied such molecular determinants recognized by selectins, galectins, and many other C-type lectins, involved in leukocyte recruitment to sites of inflammation in human tissues, lymphocyte trafficking, adhesion of human viruses to human cells, structure and immunogenicity of glycoproteins on the surfaces of human parasites. We have also explored the molecular basis of glycoconjugate biosynthesis by exploring the enzymes and molecular chaperones required for correct protein glycosylation. From these studies opportunities for translational biology have arisen, involving production of function-blocking antibodies, anti-glycan specific antibodies, and synthetic glycoconjugates, e.g. glycosulfopeptides, that specifically are recognized by GBPs. This invited short review is based in part on my presentation for the IGO Award 2019 given by the International Glycoconjugate Organization in Milan.


Assuntos
Adesão Celular/fisiologia , Açúcares/metabolismo , Animais , Galactosiltransferases/metabolismo , Glicoconjugados/metabolismo , Glicômica/métodos , Glicoproteínas/metabolismo , Glicosilação , Humanos , Inflamação/metabolismo , Lectinas/metabolismo , Polissacarídeos/metabolismo , Transdução de Sinais/fisiologia
14.
BMC Cancer ; 19(1): 588, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208374

RESUMO

BACKGROUND: Alterations in protein glycosylation patterns have potentially been targeted for biomarker discovery in a wide range of diseases including cancer. Although there have been improvements in patient diagnosis and survival for breast cancer (BC), there is no clinically validated serum biomarker for its early diagnosis. Here, we profiled whole serum and purified Immunoglobulin G (IgG) fraction N-glycome towards identification of non-invasive glycan markers of BC. METHODS: We employed a comprehensive glycomics approach by integrating glycoblotting-based glycan purification with MALDI-TOF/MS based quantitative analysis. Sera of BC patients belonging to stages I-IV and normal controls (NC) were collected from Ethiopian women during 2015-2016. IgG was purified by affinity chromatography using protein G spin plate and further subjected to glycoblotting for glycan release. Mass spectral data were further processed and evaluated rigorously, using various bioinformatics and statistical tools. RESULTS: Out of 35 N-glycans that were significantly up-regulated in the sera of all BC patients compared to the NC, 17 complex type N-glycans showed profound expression abundance and diagnostic potential (AUC = 0.8-1) for the early stage (I and II) BC patients. Most of these glycans were core-fucosylated, multiply branched and sialylated structures, whose abundance has been strongly associated with greater invasive and metastatic potential of cancer. N-glycans quantified form IgG confirmed their abundance in BC patients, of which two core-fucosylated and agalactosylated glycans (m/z 1591, 1794) could specifically distinguish (AUC = 0.944 and 0.921, p ≤ 0.001) stage II patients from NC. Abundance of such structural features in IgG is associated with a decrease in its immunosuppressive potential towards tumor cells, which in part may correlate with the aggressive nature of BC commonly noticed in black population. CONCLUSIONS: Our comprehensive study has addressed for the first time both whole serum and IgG N-glycosylation signatures of native black women suffering from BC and revealed novel glyco-biomarkers with marked overexpression and distinguishing ability at early stage patients. Further studies on direct identification of the intact glycoproteins using a glycoprteomics approach will provide a deeper understanding of specific biomarkers towards their clinical utility.


Assuntos
Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Detecção Precoce de Câncer , Imunoglobulina G/sangue , Adulto , Biomarcadores Tumorais/sangue , Etiópia , Feminino , Glicômica/métodos , Glicoproteínas/sangue , Glicosilação , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Polissacarídeos/sangue , Curva ROC , Reprodutibilidade dos Testes
15.
J Chromatogr A ; 1600: 105-111, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31056268

RESUMO

Efficient sample pretreatment of N-glycans from glycoproteins is essential but challenging due to the limitations of existing tedious and laborious methods in N-glycomics. This study aimed to establish a filter-aided extraction method coupled with glycosylamine AQC labeling for a simple and rapid direct HPLC-FLD-based analysis of N-glycans. The developed method was demonstrated to be simpler and more sensitive compared to previous HILIC SPE purification method coupled with glycosylamine labeling. It has been validated with wild-type N-glycans from human transferrin and RNase B and then was successfully applied to investigate N-glycan profiles of the transferrin in human serum and a monoclonal antibody (mAb). Results showed good applicability of the method for complex samples. Additionally, this method is compatible with the replicate determination of N-glycan samples to assess the high-throughput analysis of glycan variability in mAb sample.


Assuntos
Cromatografia Líquida de Alta Pressão , Glicômica/métodos , Polissacarídeos/análise , Filtração , Glicômica/instrumentação , Glicoproteínas , Glicosilação , Humanos , Reprodutibilidade dos Testes , Transferrina/análise
16.
Analyst ; 144(11): 3601-3612, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31065629

RESUMO

Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins. This approach is especially valuable when resolving structurally similar isomers and for discovery of novel and/or sample-specific glycan structures. However, the implementation of PGC-based separations in glycomics studies has been limited because system-independent retention values have not been established to normalize technical variation. To address this limitation, this study combined the use of hydrolyzed dextran as an internal standard and Skyline software for post-acquisition normalization to reduce retention time and peak area technical variation in PGC-based glycan analyses. This approach allowed assignment of system-independent retention values that are applicable to typical PGC-based glycan separations and supported the construction of a library containing >300 PGC-separated glycan structures with normalized glucose unit (GU) retention values. To enable the automation of this normalization method, a spectral MS/MS library was developed of the dextran ladder, achieving confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two ways. First, to inform the search space for bioinformatically predicted but unobserved glycan structures, predictive models for two structural modifications, core-fucosylation and bisecting GlcNAc, were developed based on the GU library. Second, the applicability of this method for the analysis of complex biological samples is evidenced by the ability to discriminate between cell culture and tissue sample types by the normalized intensity of N-glycan structures alone. Overall, the methods and data described here are expected to support the future development of more automated approaches to glycan identification and quantitation.


Assuntos
Cromatografia Líquida/normas , Glicômica/normas , Polissacarídeos/análise , Espectrometria de Massas em Tandem/normas , Animais , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Glicômica/métodos , Grafite/química , Células HEK293 , Humanos , Isomerismo , Masculino , Camundongos Endogâmicos BALB C , Polissacarídeos/química , Porosidade , Espectrometria de Massas em Tandem/métodos
17.
Nat Commun ; 10(1): 2137, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086181

RESUMO

The in-depth, high-sensitivity characterization of the glycome from complex biological samples, such as biofluids and tissues, is of utmost importance in basic biological research and biomarker discovery. Major challenges often arise from the vast structural diversity of glycans in combination with limited sample amounts. Here, we present a method for the highly sensitive characterization of released N-glycans by combining a capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) approach with linkage-specific derivatization of sialic acids and uniform cationic reducing end labelling of all glycans. This method allows the analysis of glycans at the attomole level, provides information on sialic acid isomers and enables the in-depth characterization of complex samples, even when available in minute amounts.


Assuntos
Glicômica/métodos , Polissacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Proteínas Sanguíneas/química , Eletroforese Capilar/métodos , Glicosilação , Humanos , Isomerismo , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Ácidos Siálicos/química
18.
Biochem Biophys Res Commun ; 513(1): 186-192, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30952424

RESUMO

O-Linked glycan liberation from proteins through reductive beta-elimination and hydrazinolysis is widely used, but have yet to satisfy the recent needs for glycan analysis in glycan biomarker research and microheterogeneity evaluation of biopharmaceutical glycosylation. Here, we introduce an alternative method by using hydroxylamine and an organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and optimize the reaction conditions. The developed method afforded comparable results to those of hydrazinolysis, but with less degraded products. In addition, we examined the compatibility of the optimized O-linked glycan liberation with denaturant and detergents. The optimized method also released glycans containing NeuGc without degradation or deacylation. To demonstrate the feasibility of the developed method, we analyzed O-linked glycans of porcine submaxillary mucins separated by supported molecular matrix electrophoresis (SMME) which is previously developed to characterize mucins. The method for O-linked glycan liberation and fluorescent labeling presented here was easy and rapid, and will be practically useful for O-linked glycan analyses.


Assuntos
Glicoproteínas/química , Hidroxilamina/química , Polissacarídeos/análise , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/métodos , Glicômica/métodos , Glicosilação , Mucinas/química , Polissacarídeos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Suínos
19.
Glycoconj J ; 36(2): 175-183, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30993518

RESUMO

Lectins, in combination with our established enzyme-linked lectin sorbent assay (ELLSA) and inhibition study, have been used as powerful tools in many glycoconjugate recognition studies. In this short review, we highlight the following: (i) The recognition profiles of Gal/GalNAc-specific lectins were updated and upgraded. (ii) Based on the cross-specificities of applied lectins, a new classification system was introduced. (iii) Applications of lectins for the detection and identification of N-glycan and/or Tn glycotope in glycoconjugates were intergraded. (iv) The polyvalency of the glycotopes in glycans was found to play a critical role in glycan-lectin recognition. This is an unexplored area of glycobiology and one of the most promising directions toward the coming glycoscience transformation.


Assuntos
Glicômica/métodos , Lectinas/metabolismo , Animais , Humanos , Lectinas/química , Técnicas de Sonda Molecular , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica
20.
Glycobiology ; 29(6): 452-460, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30913289

RESUMO

Mass spectrometry (MS) is one of the most effective techniques for high-throughput, high-resolution characterization of glycan structures. Although many software applications have been developed over the last decades for the interpretation of MS data of glycan structures, only a few are capable of dealing with the large data sets produced by glycomics analysis. Furthermore, these applications utilize databases that can lead to redundant glycan annotations and do not support post-processing of the data within the software or by third party applications. To address the needs, we present GRITS Toolbox, a freely-available, platform-independent software application capable of storing and processing glycomics MS data along with associated metadata. GRITS Toolbox automatically annotates MS data using an integrated glycan identification module that references manually curated databases of mammalian glycans (provided with the software) or any user-defined databases. Extensive display routines are provided to post-process the data and refine the automated annotation using expert knowledge of the user. The software also allows side by side comparison of annotations from different MS runs or samples and exporting of annotations into Excel format.


Assuntos
Glicômica/métodos , Espectrometria de Massas , Software , Bases de Dados Factuais
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