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1.
Glycobiology ; 27(12): 1099-1108, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28973482

RESUMO

Juvenile idiopathic arthritis (JIA) encompasses all forms of chronic idiopathic arthritis that arise before age 16. Previous studies have found JIA to be associated with lower Fc galactosylation of circulating IgG, but the overall spectrum of glycan changes and the net impact on IgG function are unknown. Using ultra performance liquid chromatography (UPLC), we compared IgG glycosylation in 54 subjects with recent-onset untreated JIA with 98 healthy pediatric controls, paired to biophysical profiling of affinity for 20 IgG receptors using a high-throughput multiplexed microsphere assay. Patients with JIA exhibited an increase in hypogalactosylated and hyposialylated IgG glycans, but no change in fucosylation or bisection, together with alteration in the spectrum of IgG ligand binding. Supervised machine learning demonstrated a robust capacity to discriminate JIA subjects from controls using either glycosylation or binding data. The binding signature was driven predominantly by enhanced affinity for Fc receptor like protein 5 (FcRL5), a noncanonical Fc receptor expressed on B cells. Affinity for FcRL5 correlated inversely with galactosylation and sialylation, a relationship confirmed through enzymatic manipulation. These results demonstrate the capacity of combined structural and biophysical IgG phenotyping to define the overall functional impact of IgG glycan changes and implicate FcRL5 as a potential cellular sensor of IgG glycosylation.


Assuntos
Artrite Juvenil , Sítios de Ligação de Anticorpos , Fragmentos Fc das Imunoglobulinas , Imunoglobulina G , Receptores Fc , Adolescente , Artrite Juvenil/sangue , Artrite Juvenil/imunologia , Criança , Pré-Escolar , Feminino , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/sangue , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Lactente , Masculino , Receptores Fc/sangue , Receptores Fc/imunologia
3.
Proteomics ; 11(17): 3582-6, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21751342

RESUMO

We have undertaken the identification of basic proteins (pH 6-11) of the human heart using 2-DE. Tissue from the left ventricle of human heart was lysed and proteins were separated in the first dimension on pH 6-11 IPG strips using paper-bridge loading followed by separation on 12% SDS polyacrylamide gels in the second dimension. Proteins were then identified by mass spectrometry and analysed using Proline, a proteomic data analysis platform that was developed in-house. The proteome map contains 176 identified spots with 151 unique proteins and has been made available as part of the UCD-2DPAGE database at http://proteomics-portal.ucd.ie:8082. The associated mass spectrometry data have been submitted to PRIDE (Accession number ♯10098). This reference map, and the other heart reference maps available through the UCD-2DPAGE database, will aid further proteomic studies of heart diseases such as dilated cardiomyopathy and ischaemic heart disease.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Miocárdio/química , Proteoma/análise , Humanos , Concentração de Íons de Hidrogênio
4.
Proteomics ; 10(13): 2551-5, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20432482

RESUMO

We describe a 2-DE proteomic reference map containing 227 basic proteins in the dorsolateral prefrontal cortex region of the human brain. Proteins were separated in the first dimension on pH 6-11 IPG strips using paper-bridge loading and on 12% SDS-PAGE in the second dimension. Proteins were subsequently identified by MS and spectra were analyzed using an in-house proteomics data analysis platform, Proline. The 2-DE reference map is available via the UCD 2-DE Proteome Database (http://proteomics-portal.ucd.ie:8082) and can also be accessed via the WORLD-2DPAGE Portal (http://www.expasy.ch/world-2dpage/). The associated protein identification data have been submitted to the PRIDE database (accession numbers 10018-10033). Separation of proteins in the basic region resolves more membrane associated proteins relevant to the synaptic pathology central to many neurological disorders. The 2-DE reference map will aid with further characterisation of neurological disorders such as bipolar and schizophrenia.


Assuntos
Química Encefálica , Córtex Pré-Frontal/química , Proteoma/análise , Eletroforese em Gel Bidimensional , Humanos
5.
Proteomics ; 9(12): 3383-94, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19562804

RESUMO

Recent studies have suggested a protective role of hsp27 against atherosclerosis and transplant graft vasculopathy. Here we have investigated the effects of over-expression of wild-type hsp27 and its phosphorylation mimics on proliferation of human endothelial cells (ECs) and smooth muscle cells (SMCs). ECs and SMCs cultured from human blood vessels or cells lines (human microvascular endothelial cell line and human telomerase reverse transcriptase subunit SMC) were infected with adenovirus containing DNA from wild-type hsp27, hyper-phosphorylated hsp27 mimic (3D hsp27), hypo-phosphorylated hsp27 mimic (3A hsp27) or anti-sense hsp27, and proliferation measured over the next 5 days. Protein extracts from infected cells were subjected to proteomic analysis using 2-D DIGE. Over-expression of 3D hsp27 and anti-sense hsp27 but not 3A hsp27 mimic caused significant inhibition of proliferation of ECs and SMCs. Proteomic analysis focussed on proteins that were significantly down-regulated by the 3D hsp27 mutant. The cell cycling proteins stathmin, cofilin and ubiquitination enzymes fullfilled these criteria. 1-D Western blots of infected human microvascular endothelial cell line and human telomerase reverse transcriptase subunit SMC confirmed down-regulation of stathmin, cofilin and ubiquitination enzymes by 3D hsp27. The phosphorylation status of hsp27 is an important regulator of proliferation of human vascular ECs and SMCs; possibly contributing to cardiovascular protection.


Assuntos
Células Endoteliais/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Músculo Liso/metabolismo , Análise de Variância , Aterosclerose , Western Blotting , Ciclo Celular , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Eletroforese em Gel Bidimensional , Células Endoteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico HSP27/biossíntese , Proteínas de Choque Térmico HSP27/genética , Humanos , Músculo Liso/citologia , Mutação , Fosforilação , Proteoma/metabolismo , Reprodutibilidade dos Testes
6.
Methods Mol Biol ; 536: 515-26, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19378088

RESUMO

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis has been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with the recently developed ECL-Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27-kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.


Assuntos
Corantes Fluorescentes/química , Immunoblotting/métodos , Proteômica/métodos , Animais , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas de Choque Térmico HSP27/análise , Humanos , Immunoblotting/instrumentação , Miocárdio/química
7.
Proteomics ; 8(20): 4160-2, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18814334

RESUMO

The workshop assembled an excellent collection of speakers from across Ireland and beyond who presented many interesting and diverse topical issues. Various proteomic applications were discussed throughout the day ranging from 2-DE and 2-D DIGE, to GeLC-MS/MS, high density Protein and Antibody Arrays, with a particular focus on the importance of quantitative mass spectrometry in proteomics.


Assuntos
Proteômica/métodos , Animais , Biomarcadores/análise , Humanos
8.
Sci Rep ; 8(1): 8655, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29872119

RESUMO

Aberrant glycosylation has been associated with a number of diseases including cancer. Our aim was to elucidate changes in whole plasma N-glycosylation between colorectal cancer (CRC) cases and controls in one of the largest cohorts of its kind. A set of 633 CRC patients and 478 age and gender matched controls was analysed. Additionally, patients were stratified into four CRC stages. Moreover, N-glycan analysis was carried out in plasma of 40 patients collected prior to the initial diagnosis of CRC. Statistically significant differences were observed in the plasma N-glycome at all stages of CRC, this included a highly significant decrease in relation to the core fucosylated bi-antennary glycans F(6)A2G2 and F(6)A2G2S(6)1 (P < 0.0009). Stage 1 showed a unique biomarker signature compared to stages 2, 3 and 4. There were indications that at risk groups could be identified from the glycome (retrospective AUC = 0.77 and prospective AUC = 0.65). N-glycome biomarkers related to the pathogenic progress of the disease would be a considerable asset in a clinical setting and it could enable novel therapeutics to be developed to target the disease in patients at risk of progression.


Assuntos
Proteínas Sanguíneas/química , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/patologia , Polissacarídeos/sangue , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Medição de Risco
9.
MAbs ; 9(8): 1349-1359, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28895795

RESUMO

Glycosylation is an important attribute of biopharmaceutical products to monitor from development through production. However, glycosylation analysis has traditionally been a time-consuming process with long sample preparation protocols and manual interpretation of the data. To address the challenges associated with glycan analysis, we developed a streamlined analytical solution that covers the entire process from sample preparation to data analysis. In this communication, we describe the complete analytical solution that begins with a simplified and fast N-linked glycan sample preparation protocol that can be completed in less than 1 hr. The sample preparation includes labelling with RapiFluor-MS tag to improve both fluorescence (FLR) and mass spectral (MS) sensitivities. Following HILIC-UPLC/FLR/MS analyses, the data are processed and a library search based on glucose units has been included to expedite the task of structural assignment. We then applied this total analytical solution to characterize the glycosylation of the NIST Reference Material mAb 8761. For this glycoprotein, we confidently identified 35 N-linked glycans and all three major classes, high mannose, complex, and hybrid, were present. The majority of the glycans were neutral and fucosylated; glycans featuring N-glycolylneuraminic acid and those with two galactoses connected via an α1,3-linkage were also identified.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Anticorpos Monoclonais/química , Glicoproteínas/química , Polissacarídeos/química , Anticorpos Monoclonais/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Corantes Fluorescentes/química , Glicoproteínas/metabolismo , Glicosilação , Humanos , Espectrometria de Massas/métodos , Polissacarídeos/metabolismo , Reprodutibilidade dos Testes , Fatores de Tempo
10.
Methods Mol Biol ; 1314: 139-49, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26139262

RESUMO

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis have been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with ECL Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27 kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.


Assuntos
Corantes Fluorescentes/química , Proteínas de Choque Térmico HSP27/análise , Immunoblotting/métodos , Imunoconjugados/química , Proteínas/análise , Proteômica/métodos , Animais , Anticorpos Monoclonais/química , Western Blotting/métodos , Carbocianinas/química , Eletroforese em Gel Bidimensional/métodos , Humanos , Focalização Isoelétrica/métodos , Fluxo de Trabalho
11.
Thromb Haemost ; 113(2): 290-304, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25413489

RESUMO

The integrin αIIbß3 on resting platelets can bind to immobilised fibrinogen resulting in platelet spreading and activation but requires activation to bind to soluble fibrinogen. αIIbß3 is known to interact with the general integrin-recognition motif RGD (arginine-glycine-aspartate) as well as the fibrinogen-specific γ-chain dodecapeptide; however, it is not known how fibrinogen binding triggers platelet activation. NGR (asparagine-glycine-arginine) is another integrin-recognition sequence present in fibrinogen and this study aims to determine if it plays a role in the interaction between fibrinogen and αIIbß3. NGR-containing peptides inhibited resting platelet adhesion to fibrinogen with an IC50 of 175 µM but failed to inhibit the adhesion of activated platelets to fibrinogen (IC50> 500 µM). Resting platelet adhesion to mutant fibrinogens lacking the NGR sequences was reduced compared to normal fibrinogen under both static and shear conditions (200 s⁻¹). However, pre-activated platelets were able to fully spread on all types of fibrinogen. Thus, the NGR motif in fibrinogen is the site that is primarily responsible for the interaction with resting αIIbß3 and is responsible for triggering platelet activation.


Assuntos
Plaquetas/fisiologia , Fibrinogênio/química , Oligopeptídeos/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Motivos de Aminoácidos , Animais , Coagulação Sanguínea , Antígenos CD13/química , Células CHO , Células COS , Adesão Celular , Chlorocebus aethiops , Cricetulus , Ensaio de Imunoadsorção Enzimática , Voluntários Saudáveis , Humanos , Concentração Inibidora 50 , Ligantes , Microscopia Confocal , Microscopia de Fluorescência , Peptídeos/química , Ativação Plaquetária , Adesividade Plaquetária , Agregação Plaquetária/fisiologia , Testes de Função Plaquetária , Glicoproteínas da Membrana de Plaquetas/química , Ligação Proteica , Proteínas Recombinantes/química
12.
Methods Mol Biol ; 899: 293-313, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22735961

RESUMO

N-linked oligosaccharides are complex non-template-derived structures that are attached to the side chains of asparagine, via the nitrogen atom. Specific changes in the N-glycans of serum glycoproteins have been associated with the pathogenesis of many diseases. The oligosaccharides present on the C(H)2 domain of immunoglobulins are known to modulate the effector functions of the molecule. These glycans provoke various biological effects, necessitating the development of robust high-throughput technology in order to fully characterize the N-glycosylation profile. This chapter describes in detail four methods to release N-glycans from the glycoprotein of interest. Two of these protocols, referred to as the "In-Gel Block" and "1D sodium dodecyl sulfate-polyacrylamide gel electrophoresis" methods, require immobilization of the glycoprotein prior to analysis. An automated method is also described, involving the purification of immunoglobulins directly from fermentation media, and, finally, an "In-solution method" is detailed, which directly releases the N-glycans into solution. HILIC and WAX-HPLC are used to analyze the N-glycan profile. Exoglycosidase enzymes digestion arrays, in combination with computer-assisted data analysis, are used to determine both the sequence and linkage of the N-glycans present.


Assuntos
Glicoproteínas , Ensaios de Triagem em Larga Escala/métodos , Biologia Molecular/métodos , Polissacarídeos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/análise , Glicoproteínas/química , Glicosilação , Humanos , Proteínas Imobilizadas/química , Imunoglobulinas/isolamento & purificação , Monossacarídeos/química , Polissacarídeos/análise , Polissacarídeos/química
13.
Proteomics ; 7(3): 332-6, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17274074

RESUMO

This report reviews the 7th Siena Meeting 'From Genome to Proteome: Back to the Future' which took place in Italy from 3-7 September, 2006. There was a significant rise in the number of delegates attending compared with previous Siena meetings. A diversity of speakers and presentations addressed the theme of the meeting in moving proteomics forward to integrate with biology as a whole entity rather than in isolated fractions. In addition, technological advancements in sample preparation and separation as well as identification were discussed.


Assuntos
Genoma , Genômica/tendências , Proteoma , Proteômica/tendências , Animais , Humanos , Comunicação Interdisciplinar , Itália
14.
Proteomics ; 6(24): 6400-4, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17111436

RESUMO

The development of ECL-Plex CyDye-conjugated secondary antibodies allows the advancement of conventional Western blotting, opening up possibilities for highly sensitive and quantitative protein confirmation and identification. We report a novel proteomic method to simultaneously visualise the total protein profile as well as the specific immunodetection of an individual protein species by combining cyanine CyDye pre-labelled proteins and antibody immunoblotting. This technique proposes to revolutionise both 2-D immunoprobing and protein confirmation following MS analysis.


Assuntos
Immunoblotting/métodos , Proteômica/métodos , Sequência de Aminoácidos , Carbocianinas , Corantes Fluorescentes , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/imunologia , Proteínas de Choque Térmico/isolamento & purificação , Humanos , Imunoquímica , Chaperonas Moleculares , Miocárdio/química , Proteínas de Neoplasias/química , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Análise Serial de Proteínas/métodos , Proteínas/imunologia , Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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