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1.
J Chromatogr A ; 1610: 460545, 2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31551124

RESUMO

The facile enrichment of glycopeptides or glycoproteins poses great challenges for glycoproteomic research. In this study, a novel hydrophilic material, named zwitterionic hydrophilic L-cysteine derivatized straticulate-C3N4 composites (LCAC), were synthesized and evaluated for the enrichment of N-glycopeptides. LCAC exhibited good biocompatibility, excellent hydrophilicity and selectivity, by virtue of the large surface of C3N4 and the zwitterionic property offered by cysteine. LCAC demonstrated excellent performance for N-glycopeptide enrichment with the sensitivity of 0.033 fmol/µL, selectivity of 1:100, and high recovery rate (∼85%). The performance of LCAC was demonstrated by the identification of 35 N-glycopeptides from IgG, as well as capturing 1809 human urine N-glycopeptides corresponding to 876 N-glycoproteins. Comparing the LCAC with our developed phenylboronic acid functionalized material showed a certain complementary due to the different binding mechanism. The simple production and enhanced hydrophilic properties make the material a promising choice for glycoproteomics researches.


Assuntos
Cisteína/química , Glicopeptídeos/isolamento & purificação , Glicoproteínas/isolamento & purificação , Nitrilos/química , Cromatografia de Afinidade , Glicopeptídeos/urina , Glicoproteínas/urina , Humanos , Interações Hidrofóbicas e Hidrofílicas , Sensibilidade e Especificidade
2.
Nanoscale ; 11(8): 3701-3709, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30742181

RESUMO

The highly effective analysis of glycopeptides from complex biological samples is an attractive and critical topic all the time. In this study, a novel thickness-controlled hydrophilic Mg-metal organic frameworks (Mg-MOFs) coating-functionalized magnetic graphene composite (MagG@Mg-MOFs-1C) was prepared for the capture of the glycopeptides. The as-synthesized composite exhibits an ultralow limit of detection (0.1 fmol µL-1), a perfect size-exclusion effect (HRP digests/BSA protein/HRP protein, 1 : 500 : 500, w/w/w), and a high binding capacity (150 mg g-1), satisfying reusability and high recovery in the recognition of glycopeptides due to its outstanding characteristics including strong magnetic property, large surface area (617 m2 g-1), plenty of affinity sites, and excellent hydrophilicity. Furthermore, the MagG@Mg-MOFs-1C composite was successfully applied to selectively enriched glycopeptides in human urine. More excitingly, 406 N-glycosylation peptides corresponding to 185 glycoproteins were identified in the urine of the bladder cancer patients, in which these identified glycoproteins include the potential biomarkers (α-2-macroglobulin, complement C4-B, and α-1-antitrypsin) for the bladder cancer. This study suggests that the hydrophilic porous MOFs-functionalized composite has a great potential in the large-scale characterization of the low-abundance biomolecules in urine, opening a new avenue for the rapid and convenient diagnosis of the disease.


Assuntos
Glicopeptídeos/urina , Grafite/química , Magnésio/química , Magnetismo , Estruturas Metalorgânicas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Cromatografia Líquida de Alta Pressão , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Ligação Proteica , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
3.
Proteomics Clin Appl ; 13(3): e1800111, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30334612

RESUMO

PURPOSE: Urine is a rich source of potential biomarkers, including glycoproteins. Glycoproteomic analysis remains difficult due to the high heterogeneity of glycans. Nevertheless, recent advances in glycoproteomics software solutions facilitate glycopeptide identification and characterization. The aim is to investigate intact glycopeptides in the urinary peptide profiles of normal subjects using a novel PTM-centric software-Byonic. EXPERIMENTAL DESIGN: The urinary peptide profiles of 238 normal subjects, previously analyzed using CE-MS and CE-MS/MS and/or LC-MS/MS, are subjected to glycopeptide analysis. Additionally, glycopeptide distribution is assessed in a set of 969 patients with five different cancer types: bladder, prostate and pancreatic cancer, cholangiocarcinoma, and renal cell carcinoma. RESULTS: A total of 37 intact O-glycopeptides and 23 intact N-glycopeptides are identified in the urinary profiles of 238 normal subjects. Among the most commonly identified O-glycoproteins are Apolipoprotein C-III and insulin-like growth factor II, while titin among the N-glycoproteins. Further statistical analysis reveals that three O-glycopeptides and five N-glycopeptides differed significantly in their abundance among the different cancer types, comparing to normal subjects. CONCLUSIONS AND CLINICAL RELEVANCE: Through the established glycoproteomics workflow, intact O- and N-glycopeptides in human urine are identified and characterized, providing novel insights for further exploration of the glycoproteome with respect to specific diseases.


Assuntos
Glicopeptídeos/urina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/urina , Biomarcadores/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/urina , Proteômica , Software , Adulto Jovem
4.
Talanta ; 191: 509-518, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30262092

RESUMO

It is challenging to capture N-glycopeptides with high recovery and high specificity from complicated biosystems. Herein, we present a facile and economical procedure to generate a novel self-assembling 4-Mercaptobenzene boronic acid functionalized and Au-doped Straticulate C3N4 (MASC), with enhanced affinity capability towards glycopeptides. The materials possess low pH value adaptation, high hydrophilicity and stability, good repeatability and recyclability, and provided high selectivity (1:100), low limit of detection (0.33 fmol/µL), high enrichment efficiency (~ 80%) and high recovery rate (~ 90%) towards glycopeptides. The materials can capture glycopeptides unbiasedly, as demonstrated by the identification of 37 glycopeptides from IgG and 21 glycopeptides from horseradish peroxidase (HRP). The performance of MASC on human urine and serum glycoproteome analysis was also tested. An average of 1465 glycopeptides from 839 glycoproteins and 1553 glycopeptides from 884 glycoproteins were identified from female and male urine samples in a single mass spectrometry analysis. O-glycopeptides from human urine were also significantly enriched. Additionally, 463 glycopeptides assigned to 209 glycoproteins were identified from 5 µL of human serum. All of these results indicate that MASC presents a good performance and applicability in the field of glycoproteomic research.


Assuntos
Ácidos Borônicos/química , Glicopeptídeos/química , Interações Hidrofóbicas e Hidrofílicas , Nitrilos/química , Feminino , Glicopeptídeos/metabolismo , Glicopeptídeos/urina , Humanos , Masculino , Modelos Moleculares , Conformação Molecular , Caracteres Sexuais , Tripsina/metabolismo
5.
ACS Infect Dis ; 5(3): 353-364, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30585483

RESUMO

The evaluation of new tuberculosis (TB) therapies is limited by the paucity of biomarkers to monitor treatment response. Previous work detected an uncharacterized urine metabolite with a molecular mass of 874.3547 Da that showed promise as a biomarker for successful TB treatment. Using mass spectrometry combined with enzymatic digestions, the metabolite was structurally characterized as a seryl-leucine core 1 O-glycosylated peptide (SLC1G) of human origin. Examination of SLC1G in urine revealed a significant abundance increase in individuals with active TB versus their household contacts and healthy controls. Moreover, differential decreases in SLC1G levels were observed by week one in TB patients during successful treatment versus those that failed treatment. The SLC1G levels were also associated with clinical parameters used to measure bacterial burden (GeneXpert) and inflammation (positron emission tomography-computed tomography (PET-CT)). These results demonstrate the importance of metabolite identification and provide strong evidence for applying SLC1G as a biomarker of TB treatment response.


Assuntos
Antituberculosos/uso terapêutico , Glicopeptídeos/urina , Tuberculose/tratamento farmacológico , Adulto , Biomarcadores/urina , Monitoramento de Medicamentos , Feminino , Humanos , Leucina , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Tuberculose/urina , Urina/química , Adulto Jovem
6.
Anal Bioanal Chem ; 410(28): 7305-7312, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30171281

RESUMO

Urine is an attractive and non-invasive alternative source to tissue, blood or other biofluids for biomarker screening in clinical research. In normal human adult urine, 48% of the total urinary protein is in the sediment, 49% is soluble and the remaining 3% is contained in urinary extracellular vesicles (EVs). The soluble proteins and EV proteins in urine have attracted particular attention in recent years as cancer diagnostics. Furthermore, considering the important role of N-glycoproteins in practically all physiological processes, including regulating receptor-ligand binding, cell-cell interactions, inflammatory response and tumour progression, N-glycoproteome in human urine is an invaluable target for monitoring the physiological status and pathological changes of the kidney and urinary tract. Given the different origins of the soluble proteins and EV proteins in the urine, different N-glycoproteome patterns exist. Therefore, isolating the soluble N-glycoproteins and EV N-glycoproteins for separate analysis will provide a more specific and comprehensive view and provide a deeper understanding of human urinary N-glycoproteome. In this work, we developed a sequential separation method that isolates urinary soluble proteins and EV proteins via stepwise ultrafiltration based on their obvious size difference. A facile and reproducible protein isolation was achieved using this strategy. Subsequent N-glycoproteome enrichment and identification revealed distinct patterns in the two sub-proteomes of urine with more than 60% differential N-glycopeptides. A more comprehensive picture of the urinary N-glycoproteome with close to 1800 identified N-glycopeptides was obtained by this new analysis strategy, therefore making it advantageous for urinary biomarker screening. Graphical abstract A sequential separation method that isolates urinary soluble proteins and EV proteins via stepwise ultrafiltration was developed in this work. Subsequent N-glycopeptides enrichment and mass spectrometry analysis reveals distinct N-glycoproteome patterns in the two sub-proteomes of urine and a deep mapping of close to 1800 N-glycopeptides.


Assuntos
Glicopeptídeos/química , Glicopeptídeos/urina , Proteoma , Ultrafiltração/métodos , Adulto , Feminino , Humanos , Masculino , Proteinúria
7.
J Am Soc Mass Spectrom ; 29(6): 1210-1220, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29730764

RESUMO

A very complex mixture of intact, human N- and O-glycopeptides, enriched from the tryptic digest of urinary proteins of three healthy donors using a two-step lectin affinity enrichment, was analyzed by LC-MS/MS, leading to approximately 45,000 glycopeptide EThcD spectra. Two search engines, Byonic and Protein Prospector, were used for the interpretation of the data, and N- and O-linked glycopeptides were assigned from separate searches. The identification rate was very low in all searches, even when results were combined. Thus, we investigated the reasons why was it so, to help to improve the identification success rate. Focusing on O-linked glycopeptides, we noticed that in EThcD, larger glycan oxonium ions better survive the activation than those in HCD. These fragments, combined with reducing terminal Y ions, provide important information about the glycan(s) present, so we investigated whether filtering the peaklists for glycan oxonium ions indicating the presence of a tetra- or hexasaccharide structure would help to reveal all molecules containing such glycans. Our study showed that intact glycans frequently do not survive even mild supplemental activation, meaning one cannot rely on these oxonium ions exclusively. We found that ETD efficiency is still a limiting factor, and for highly glycosylated peptides, the only information revealed in EThcD was related to the glycan structures. The limited overlap of results delivered by the two search engines draws attention to the fact that automated data interpretation of O-linked glycopeptides is not even close to being solved. Graphical abstract ᅟ.


Assuntos
Glicopeptídeos/urina , Espectrometria de Massas em Tandem/métodos , Adulto , Sequência de Carboidratos , Cromatografia Líquida/métodos , Feminino , Glicopeptídeos/análise , Humanos , Masculino , Pessoa de Meia-Idade , Ferramenta de Busca
8.
Eur J Nutr ; 57(5): 1883-1890, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28578535

RESUMO

PURPOSE: Inter-individual variation in median plasma copeptin is associated with incident type 2 diabetes mellitus, progression of chronic kidney disease, and cardiovascular events. In this study, we examined whether 24-h urine osmolality was associated with plasma copeptin and whether increasing daily water intake could impact circulating plasma copeptin. METHODS: This trial was a prospective study conducted at a single investigating center. Eighty-two healthy adults (age 23.6 ± 2.9 years, BMI 22.2 ± 1.5 kg/m2, 50% female) were stratified based upon habitual daily fluid intake volumes: arm A (50-80% of EFSA dietary reference values), arm B (81-120%), and arm C (121-200%). Following a baseline visit, arms A and B increased their water intake to match arm C for a period of 6 consecutive weeks. RESULTS: At baseline, plasma copeptin was positively and significantly associated with 24-h urine osmolality (p = 0.002) and 24-h urine specific gravity (p = 0.003) but not with plasma osmolality (p = 0.18), 24-h urine creatinine (p = 0.09), and total fluid intake (p = 0.52). Over the 6-week follow-up, copeptin decreased significantly from 5.18 (3.3;7.4) to 3.90 (2.7;5.7) pmol/L (p = 0.012), while urine osmolality and urine specific gravity decreased from 591 ± 206 to 364 ± 117 mOsm/kg (p < 0.001) and from 1.016 ± 0.005 to 1.010 ± 0.004 (p < 0.001), respectively. CONCLUSIONS: At baseline, circulating levels of copeptin were positively associated with 24-h urine concentration in healthy young subjects with various fluid intakes. Moreover, this study shows, for the first time, that increased water intake over 6 weeks results in an attenuation of circulating copeptin. CLINICAL TRIAL REGISTRATION NUMBER: NCT02044679.


Assuntos
Ingestão de Líquidos , Glicopeptídeos/sangue , Glicopeptídeos/urina , Concentração Osmolar , Urinálise , Adulto , Feminino , França , Humanos , Masculino , Estudos Prospectivos
9.
PLoS One ; 12(1): e0169263, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28081165

RESUMO

BACKGROUND: Autosomal Dominant Polycystic Kidney Disease (ADPKD) patients have an impaired urine concentrating capacity. Increased circulating vasopressin (AVP) concentrations are supposed to play a role in the progression of ADPKD. We hypothesized that ADPKD patients have a more severely impaired urine concentrating capacity in comparison to other patients with chronic kidney disease at a similar level of kidney function, with consequently an enhanced AVP response to water deprivation with higher circulating AVP concentrations. METHODS: 15 ADPKD (eGFR<60) patients and 15 age-, sex- and eGFR-matched controls with IgA nephropathy (IgAN), underwent a water deprivation test to determine maximal urine concentrating capacity. Plasma and urine osmolality, urine aquaporin-2 (AQP2) and plasma AVP and copeptin (a surrogate marker for AVP) were measured at baseline and after water deprivation (average 16 hours). In ADPKD patients, height adjusted total kidney volume (hTKV) was measured by MRI. RESULTS: Maximal achieved urine concentration was lower in ADPKD compared to IgAN controls (533±138 vs. 642±148 mOsm/kg, p = 0.046), with particularly a lower maximal achieved urine urea concentration (223±74 vs. 299±72 mmol/L, p = 0.008). After water deprivation, plasma osmolality was similar in both groups although change in plasma osmolality was more profound in ADPKD due to a lower baseline plasma osmolality in comparison to IgAN controls. Copeptin and AVP increased significantly in a similar way in both groups. AVP, copeptin and urine AQP2 were inversely associated with maximal urine concentrating in both groups. CONCLUSIONS: ADPKD patients have a more severely impaired maximal urine concentrating capacity with a lower maximal achieved urine urea concentration in comparison to IgAN controls with similar endogenous copeptin and AVP responses.


Assuntos
Glomerulonefrite por IGA/urina , Glicopeptídeos/urina , Capacidade de Concentração Renal , Rim Policístico Autossômico Dominante/urina , Vasopressinas/urina , Adolescente , Adulto , Idoso , Aquaporina 2/urina , Feminino , Glomerulonefrite por IGA/diagnóstico por imagem , Humanos , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Rim Policístico Autossômico Dominante/diagnóstico por imagem
10.
Glycoconj J ; 33(6): 937-951, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27234710

RESUMO

Glycosylation is a very important post-translational modification involved in various cellular processes, such as cell adhesion, signal transduction and immune response. Urine is a rich source of glycoproteins and attractive biological fluid for biomarker discovery, owing to its availability, ease of collection, and correlation with pathophysiology of diseases. Although the urinary proteomics have been explored previously, the urinary glycoproteome characterization remains challenging requiring the development and optimization of analytical and bioinformatics methods for protein glycoprofiling. This study describes the high confident identification of 472 unique N-glycosylation sites covering 256 urinary glycoproteins. Besides, 202 unique N-glycosylation sites were identified in low molecular weight endogenous glycopeptides, which belong to 90 glycoproteins. Global site-specific characterization of the N-linked glycan heterogeneity was achieved by intact glycopeptide analysis, revealing 303 unique glycopeptides most of them displaying complex/hybrid glycans composed by sialic acid and fucose. These datasets consist in a valuable resource of glycoproteins and N-glycosylation sites found in healthy human urine that can be further explored in different disorders, in which the N-linked glycosylation may be aberrant.


Assuntos
Glicopeptídeos/urina , Glicoproteínas/urina , Adulto , Glicosilação , Humanos , Masculino
11.
Arch Physiol Biochem ; 122(3): 111-6, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26849673

RESUMO

PURPOSE: Endometrial (ECa), ovarian (OCa) and cervical (CCa) cancers are among 10 of the most common cancers affecting women worldwide. Cancers are known to cause some proteins to be differentially glycosylated or aberrantly excreted in the urine, which can be used as biomarkers. Since ECa, OCa and CCa are difficult to diagnose at the early stage, the aim of the present study was to identify a panel of new biomarkers for early detection of the cancers using surface-enhanced laser desorption/ionization-time-of-flight (SELDI-TOF) technology. Identification of early biomarkers that are specific and efficient can increase the survival rate of the patients. EXPERIMENTAL DESIGN: Digested urinary proteins from patients with ECa, OCa and CCa were incubated on the champedak mannose-binding (CMB) lectin-immobilized PS10 chip. The lectin-captured glycopeptides were detected with SELDI-TOF mass spectrometry and followed by biomarker wizard analysis. RESULTS: Peaks m/z 1201 and 1449 were detected as potential group discriminators. The peak m/z 1201 could distinguish OCa from CCa and ECa and its sensitivity and specificity were 100%. For m/z 1449, it was able to differentiate ECa from the other two types of cancer. CONCLUSIONS: The findings of this study suggest urinary glycopeptides m/z 1201 and 1449 may serve as potential biomarkers for the early detection of ECa, OCa and CCa, although this requires further extensive validation on clinically representative populations.


Assuntos
Biomarcadores Tumorais/urina , Neoplasias do Endométrio/diagnóstico , Glicopeptídeos/urina , Glicoproteínas/urina , Neoplasias Ovarianas/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neoplasias do Colo do Útero/diagnóstico , Adulto , Idoso , Estudos de Casos e Controles , Neoplasias do Endométrio/urina , Feminino , Seguimentos , Glicosilação , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/urina , Prognóstico , Análise Serial de Proteínas , Proteômica , Neoplasias do Colo do Útero/urina
12.
Rapid Commun Mass Spectrom ; 29(21): 1929-37, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26443390

RESUMO

RATIONALE: Schindler disease is caused by the deficient activity of α-N-acetylgalactosaminidase, which leads to an abnormal accumulation of O-glycopeptides in tissues and body fluids. In this work the Schindler condition is for the first time approached by ion mobility (IMS) tandem mass spectrometry (MS/MS), for determining urine glycopeptide fingerprints and discriminate isomeric structures. METHODS: IMS-MS experiments were conducted on a Synapt G2s mass spectrometer operating in negative ion mode. A glycopeptide mixture extracted from the urine of a patient suffering from Schindler disease was dissolved in methanol and infused into the mass spectrometer by electrospray ionization using a syringe-pump system. MS/MS was performed by collision-induced dissociation (CID) at low energies, after mobility separation in the transfer cell. Data acquisition and processing were performed using MassLynx and Waters Driftscope software. RESULTS: IMS-MS data indicated that the attachment of one or two amino acids to the carbohydrate backbone has a minimal influence on the molecule conformation, which limits the discrimination of the free oligosaccharides from the glycosylated amino acids and dipeptides. The structural analysis by CID MS/MS in combination with IMS-MS of species exhibiting the same m/z but different configurations demonstrated for the first time the presence of positional isomers for some of the Schindler disease biomarker candidates. CONCLUSIONS: The IMS-MS and CID MS/MS platform was for the first time optimized and applied to Schindler disease glycourinome. By this approach the separation and characterization of Neu5Ac positional isomers was possible. IMS CID MS/MS showed the ability to determine the type of the glycopeptide isomers from a series of possible candidates.


Assuntos
Glicopeptídeos/química , Glicopeptídeos/urina , Doenças por Armazenamento dos Lisossomos/urina , Distrofias Neuroaxonais/urina , Espectrometria de Massas em Tandem/métodos , alfa-N-Acetilgalactosaminidase/deficiência , Pré-Escolar , Humanos , Isomerismo , Masculino , alfa-N-Acetilgalactosaminidase/urina
13.
Clin Exp Nephrol ; 19(6): 1199-205, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25715868

RESUMO

BACKGROUND: Experimental studies suggest a detrimental role for cyclic adenosine monophosphate (cAMP) and vasopressin in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). It is unknown, however, whether urinary cAMP and copeptin concentration are associated with disease severity in patients with ADPKD. METHODS: Urinary cAMP (u-cAMP) and copeptin concentration (u-copeptin) were measured by immunoassay in ADPKD patients with CKD stage ≤4. We compared our measurements with clinical parameters including estimated glomerular filtration rate (eGFR), total kidney volume (TKV), and height-adjusted TKV (htTKV). Logarithmic transformation of all variables was performed to fulfill the requirement of equal distribution of the residuals. RESULTS: We included 50 patients in this study (24 females and 26 males; mean age: 49.3 years). The median eGFR and TKV were 53.2 ml/min/1.73 m(2) (interquartile range: IQR; 29.4-68.45) and 1138.1 ml (IQR; 814.7-2065.0), respectively. The median u-copeptin level was 12.19 (IQR; 6.91-22.32) ng/ml. Although u-cAMP/u-Cr was not significantly correlated with TKV (R = -0.006, p = 0.967) and eGFR (R = 0.077, p = 0.602), urinary copeptin/u-Cr was statistically associated with the various markers of disease severity in ADPKD [positively with TKV (R = 0.351, p = 0.014), htTKV (R = 0.383, p = 0.008) and negatively with eGFR (R = -0.304, p = 0.036)]. CONCLUSIONS: In ADPKD subjects, a higher u-copeptin is associated with disease progression, suggesting that u-copeptin may be a new surrogate marker to predict renal prognosis in ADPKD.


Assuntos
Arginina Vasopressina/metabolismo , Glicopeptídeos/urina , Rim Policístico Autossômico Dominante/metabolismo , Adulto , Idoso , Biomarcadores , Índice de Massa Corporal , AMP Cíclico/urina , Progressão da Doença , Feminino , Taxa de Filtração Glomerular , Humanos , Rim/patologia , Masculino , Pessoa de Meia-Idade , Rim Policístico Autossômico Dominante/urina , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/urina , Reprodutibilidade dos Testes
14.
Mol Cell Proteomics ; 14(1): 41-9, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25326458

RESUMO

Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.


Assuntos
alfa-Globulinas , Proteoglicanas de Sulfatos de Condroitina , Glicopeptídeos , alfa-Globulinas/líquido cefalorraquidiano , alfa-Globulinas/química , alfa-Globulinas/metabolismo , alfa-Globulinas/urina , Colecistocinina/análise , Proteoglicanas de Sulfatos de Condroitina/líquido cefalorraquidiano , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteoglicanas de Sulfatos de Condroitina/urina , Cromogranina A/análise , Cromogranina B/análise , Cromograninas/análise , Glicopeptídeos/líquido cefalorraquidiano , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicopeptídeos/urina , Humanos , Masculino , Neuropeptídeos/análise
15.
Mol Cell Proteomics ; 14(2): 263-76, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25452312

RESUMO

Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes.


Assuntos
Exossomos/metabolismo , Glicômica/métodos , Glicoproteínas/urina , Proteômica/métodos , Adulto , Sequência de Aminoácidos , Feminino , Ontologia Genética , Glicopeptídeos/química , Glicopeptídeos/urina , Glicosilação , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Polissacarídeos/química , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares
16.
Intensive Care Med ; 41(1): 68-76, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25465906

RESUMO

PURPOSE: Oliguria is a common symptom in critically ill patients and puts patients in a high risk category for further worsening renal function (WRF). We performed this study to explore the predictive value of biomarkers to predict WRF in oliguric intensive care unit (ICU) patients. PATIENTS AND METHODS: Single-center prospective observational study. ICU patients were included when they presented a first episode of oliguria. Plasma and urine biomarkers were measured: plasma and urine neutrophil gelatinase-associated lipocalin (pNGAL and uNGAL), urine α1-microglobulin, urine γ-glutamyl transferase, urine indices of tubular function, cystatin C, C terminal fragment of pro-arginine vasopressin (CT-ProAVP), and proadrenomedullin (MR-ProADM). RESULTS: One hundred eleven patients formed the cohort, of whom 41 [corrected] had worsening renal function. Simplified Acute Physiology Score (SAPS) II was 41 (31-51). WRF was associated with increased mortality (hazard ratio 8.65 [95 % confidence interval (CI) 3.0-24.9], p = 0.0002). pNGAL, MR-ProADM, and cystatin C had the best odds ratio and area under the receiver-operating characteristic curve (AUC-ROC: 0.83 [0.75-0.9], 0.82 [0.71-0.91], and 0.83 [0.74-0.90]), but not different from serum creatinine (Screat, 0.80 [0.70-0.88]). A clinical model that included age, sepsis, SAPS II, and Screat had AUC-ROC of 0.79 [0.69-0.87]; inclusion of pNGAL increased the AUC-ROC to 0.86 (p = 0.03). The category-free net reclassification index improved with pNGAL (total net reclassification index for events to higher risk 61 % and nonevents to lower 82 %). CONCLUSIONS: All episodes of oliguria do not carry the same risk. No biomarker further improved prediction of WRF compared with Screat in this selected cohort of patients at increased risk defined by oliguria.


Assuntos
Oligúria/sangue , Oligúria/urina , Insuficiência Renal/sangue , Insuficiência Renal/urina , Proteínas da Fase Aguda/urina , Adrenomedulina/urina , Idoso , alfa-Globulinas/urina , Biomarcadores/sangue , Biomarcadores/urina , Cistatina C/urina , Progressão da Doença , Feminino , Glicopeptídeos/urina , Humanos , Unidades de Terapia Intensiva , Testes de Função Renal , Lipocalina-2 , Lipocalinas/sangue , Lipocalinas/urina , Masculino , Pessoa de Meia-Idade , Escores de Disfunção Orgânica , Valor Preditivo dos Testes , Estudos Prospectivos , Precursores de Proteínas/urina , Proteínas Proto-Oncogênicas/sangue , Proteínas Proto-Oncogênicas/urina , Insuficiência Renal/terapia , gama-Glutamiltransferase/sangue , gama-Glutamiltransferase/urina
17.
Carbohydr Res ; 398: 90-100, 2014 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-25243357

RESUMO

In this study an integrative mass spectrometry (MS) approach based on fully automated chip-nanoelectrospray quadrupole time-of-flight was optimized and applied for the discovery and structural characterization of O-glycopeptides in a fraction from the urine of a patient diagnosed with Schindler disease type I. A mixture of O-glycopeptides extracted and purified from an age matched healthy subject served as the control. 49 glycoforms were discovered in the investigated urine fraction from Schindler disease versus only 14 in control urine. Structures with relevant biological significance, previously not described, such as O-fucosylated tetrasaccharides and chains up to pentadecamers O-linked to serine, threonine, or threonine-proline were identified in the pathological urine and characterized by tandem MS (MS/MS). A number of 29 species discovered here, most of which with long chain glycans, were not previously reported as associated to this condition. All glycopeptides were detected in only 1 min analysis time, with a sample consumption situated in the femtomole range.


Assuntos
Glicopeptídeos/urina , Doenças por Armazenamento dos Lisossomos/urina , Distrofias Neuroaxonais/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , alfa-N-Acetilgalactosaminidase/deficiência , Automação , Sequência de Carboidratos , Criança , Glicopeptídeos/química , Humanos , Dados de Sequência Molecular , Nanotecnologia/métodos , alfa-N-Acetilgalactosaminidase/urina
18.
Hypertension ; 61(3): 716-22, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23266541

RESUMO

Although elevated renin-angiotensin system activity and angiotensinergic signaling within the brain are required for hypertension, polydipsia, and increased metabolic rate induced by deoxycorticosterone acetate (DOCA)-salt, the contribution of specific receptor subtypes and brain nuclei mediating these responses remains poorly defined. We hypothesized that angiotensin type 1a receptors (AT(1a)R) within the subfornical organ (SFO) mediate these responses. Transgenic mice carrying a conditional allele of the endogenous AT(1a)R (AT(1a)R(flox)) were administered an adenovirus encoding Cre-recombinase and enhanced green fluorescent protein (eGFP) or adenovirus encoding eGFP alone into the lateral cerebral ventricle. Adenovirus encoding Cre-recombinase reduced AT(1a)R mRNA and induced recombination in AT(1a)R(flox) genomic DNA specifically in the SFO, without significant effect in the paraventricular or arcuate nuclei, and also induced SFO-specific recombination in ROSA(TdTomato) reporter mice. The effect of SFO-targeted ablation of endogenous AT(1a)R was evaluated in AT(1a)R(flox) mice at 3 time points: (1) baseline, (2) 1 week after virus injection but before DOCA-salt, and (3) after 3 weeks of DOCA-salt. DOCA-salt-treated mice with deletion of AT(1a)R in SFO exhibited a blunted increase in arterial pressure. Increased sympathetic cardiac modulation and urine copeptin, a marker of vasopressin release, were both significantly reduced in DOCA-salt mice when AT(1a)R was deleted in the SFO. Additionally, deletion of AT(1a)R in the SFO significantly attenuated the polydipsia, polyuria, and sodium intake in response to DOCA-salt. Together, these data highlight the contribution of AT(1a)R in the SFO to arterial pressure regulation potentially through changes on sympathetic cardiac modulation, vasopressin release, and hydromineral balance in the DOCA-salt model of hypertension.


Assuntos
Desoxicorticosterona/efeitos adversos , Hipertensão/induzido quimicamente , Mineralocorticoides/efeitos adversos , Receptor Tipo 1 de Angiotensina/fisiologia , Órgão Subfornical/efeitos dos fármacos , Órgão Subfornical/fisiopatologia , Animais , Pressão Arterial/efeitos dos fármacos , Biomarcadores/urina , Glicopeptídeos/urina , Coração/efeitos dos fármacos , Coração/inervação , Masculino , Camundongos , Camundongos Transgênicos , Polidipsia/induzido quimicamente , Poliúria/induzido quimicamente , Receptor Tipo 1 de Angiotensina/genética , Recombinação Genética , Sódio/metabolismo , Sistema Nervoso Simpático/efeitos dos fármacos
19.
J Chromatogr A ; 1224: 70-8, 2012 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-22226559

RESUMO

Purification of glycopeptides prior to the analysis by mass spectrometry (MS) is demanded due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among various purification methods, hydrophilic interaction liquid chromatography (HILIC) has become a popular method in recent years. In this work, we reported a novel magnetic bead-based zwitterionic HILIC (ZIC-HILIC) material which was fabricated by coating a zwitterionic polymer synthesized by spontaneous acid-catalyzed polymerization of 4-vinyl-pyridinium ethanesulfonate monomer on iron oxide magnetic nanoparticles. The resulting magnetic ZIC-HILIC nanoparticles were shown to provide high specificity and high recovery yield (95-100%) for the enrichment of glycopeptides from a standard glycoprotein, fetuin, using a simple magnetic bar. In addition, we proposed a two-step HILIC enrichment strategy using magnetic ZIC-HILIC nanoparticles for a large scale analysis of glycoproteins in complex biological samples. Using this approach, we identified 85 N-glycosylation sites in 53 glycoproteins from urine samples. Two novel glycosylation sites on N513 of uromodulin and N470 of lysosomal alpha-glucosidase which have not yet been reported were identified by two-step HILIC approach. Furthermore, all these identified sites were confirmed by studies conducted using PNGase F deglycosylation and 18O enzymatic labeling.


Assuntos
Cromatografia Líquida/métodos , Glicopeptídeos/isolamento & purificação , Nanopartículas de Magnetita/química , Animais , Bovinos , Fetuínas/metabolismo , Glicopeptídeos/urina , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Marcação por Isótopo , Solubilidade
20.
Mol Cell Proteomics ; 11(4): M111.013649, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22171320

RESUMO

Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS(2), Hex(5)HexNAc(4)-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e.g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS(3), of selected fragment-ions from the CID-MS(2) analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan- and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where "intact" glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt.


Assuntos
Glicopeptídeos/urina , Glicoproteínas/urina , Proteômica/métodos , Cromatografia Líquida , Diálise , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Humanos , Masculino , Ácido N-Acetilneuramínico/química , Espectrometria de Massas em Tandem
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