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1.
Childs Nerv Syst ; 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32617710

RESUMO

BACKGROUND: Many efforts have been performed in the last decade to accomplish the genomic and proteomic characterization of pediatric adamantinomatous craniopharyngioma with the purpose to elucidate the molecular mechanisms underlying the onset and development of this pediatric brain tumor, its high recurrence rate, and, although classified as a histologically benign neoplasm, its aggressive behavior. METHODS: The focus of this review is to perform the new comparison of the proteomic profiles of the solid component and the intracystic fluid of adamantinomatous craniopharyngioma based on our previous results, obtained by both the top-down and the bottom-up proteomic approaches, to disclose differences and similarities, and to discuss the results in the context of the most recent literature. RESULTS AND CONCLUSIONS: Proteins and peptides identified in the cyst fluid and in the solid component of adamantinomatous craniopharyngioma (AC) include beyond markers of inflammation (i.e., alpha-defensins), proteins involved in cell migration and protein degradation (i.e., beta-thymosin and ubiquitin peptides), whose main role might be in tumor growth and infiltration of the surrounding neural structures. These last appeared different in the solid components compared with the cyst fluid, missing their terminal part in the solid tissue, a feature generally associated to malignancies, which might represent a distinct molecular site for an aggressive behavior of AC.

2.
Artigo em Inglês | MEDLINE | ID: mdl-31202182

RESUMO

The multifactorial nature of Late Onset Alzheimer's Disease (LOAD), the AD form of major relevance on epidemiological and social aspects, has driven the original investigation by LC-MS and top-down proteomics approach of the protein repertoire of the brain tissue of TgCRND8 model mice fed with a diet deficient in B vitamins. The analysis of the acid-soluble fraction of brain tissue homogenates identified a list of proteins and peptides, proteoforms and PTMs. In order to disclose possible modulations, their relative quantification in wild type and AD model mice under both B vitamin deficient and control diets was performed. The levels of metallothionein III, guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 and brain acid soluble protein 1 showed statistically significant alterations depending on genotype, diet or both effects, respectively. Particularly, metallothionein III exhibited increased levels in TgCRND8 mice under B vitamin deficient diet with respect to wild type mice under both diets. Brain acid soluble protein 1 showed the opposite, revealing decreased levels in all diet groups of AD model mice with respect to wild type mice in control diet. Lower levels of brain acid soluble protein 1 were also observed in wild type mice under deficiency of B vitamins. These results, besides contributing to increase the knowledge of AD at molecular level, give new suggestions for deeply investigating metallothionein III and brain acid soluble protein 1 in AD.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Hiper-Homocisteinemia/metabolismo , Proteoma/metabolismo , Complexo Vitamínico B/análise , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Química Encefálica , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Cromatografia Líquida , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Hiper-Homocisteinemia/etiologia , Hiper-Homocisteinemia/genética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteoma/química , Proteoma/genética , Complexo Vitamínico B/metabolismo
3.
Dis Markers ; 2019: 3609789, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31191748

RESUMO

Although histologically benign, adamantinomatous craniopharyngioma (AC) pediatric brain tumor is a locally aggressive disease that frequently determines symptoms and hormonal dysfunctions related to the mass effect on the surrounding structures. Another typical feature of this benign neoplasm is the presence of voluminous liquid cysts frequently associated with the solid component. Even if studies have been devoted to the proteomic characterization of the tumor intracystic fluid, poor explorations have been performed on its solid part, principally investigated by transcriptomics technologies. In the present study, seven specimens of AC whole tumor tissue have been analyzed by LC-MS for a preliminary assessment of the proteomic profile by a top-down/bottom-up integrated approach. Thymosin beta 4, ubiquitin, calmodulin, S100 proteins, prothymosin α isoform 2, alpha-defensins 1-4, and fragments largely belonging to vimentin, hemoglobin, and glial fibrillary acidic protein characterized the intact proteome. The identification of alpha-defensins, formerly characterized in AC intracystic fluid, reinforces the hypothesis of a role for inflammation in tumor pathogenesis. A total number of 1798 unique elements were identified by a bottom-up approach with a special focus on the 433 proteins commonly characterized in the 85.7% of the samples analyzed. Their gene ontology classification evidenced the involvement of the adherence system, intermediate filaments, and actin cytoskeleton in tumor pathogenesis and of elements part of the Wnt, FGF, and EGFR signaling pathways. In addition, proteins involved in calcium modulation, innate immunity, inflammation, CCKR and integrin signaling, and gonadotropin-releasing hormone receptor pathways were also outlined. Further than confirming proteomic data previously obtained on AC intracystic fluid, these results offer a preliminary overview of the AC whole tissue protein phenotype, adding new hints towards the comprehension of this still obscure pediatric brain tumor.


Assuntos
Neoplasias Encefálicas/metabolismo , Craniofaringioma/metabolismo , Proteoma/metabolismo , Adolescente , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Criança , Craniofaringioma/genética , Craniofaringioma/patologia , Feminino , Humanos , Masculino , Proteoma/genética
4.
Int J Anal Chem ; 2018: 5673186, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30538747

RESUMO

A reverse phase high performance liquid chromatographic (RP-HPLC) method was developed for identification and estimation of 18-ß-glycyrrhetinic acid (GA) in HepG2 cell line. The analysis was carried out using a JASCO HPLC system with a C-18 (3 µm) Supelco reversed phase column (150 x 4.7 mm) using a mobile phase of 80% CH3OH and 20% of CH3CN: tetrahydrofuran: water (10:80:10, v/v/v). The method was linear in the concentration range of 1.5-120 µg /mL (n = 5). The LOD and LOQ were determined based on standard deviation of the y-intercept and the slope of the calibration curve. The LOD and LOQ values were found to be 11.46 µg/mL and 34.72 µg/mL, respectively. The mean percentage recovery by standard addition experiments of GA is 92.4 % ± 5.2%. The intracellular GA concentration value, obtained as mean of five different determinations, was 45.8 ± 7.45 µg/mL. We have developed a HPLC-UV method for quantitative determination of GA inside cells, with advantages in the cost reduction and economy of the analytical process.

5.
Expert Opin Biol Ther ; 15 Suppl 1: S191-201, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26095945

RESUMO

OBJECTIVES: The aim of this study was to characterize ß and α thymosins and their proteoforms in various tissues and bodily fluids by mass spectrometry and to look at their association with a wide variety of pathologies. METHODS: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides. RESULTS: In addition to thymosin ß4 (Tß4) and ß10 (Tß10), several post-translational modifications of both these peptides were identified not only in bodily fluids but also in normal and pathological tissues of different origins. The analysis of tissue specimens allowed the characterization of different C-terminal truncated forms of Tß4 and Tß10 together with other proteolytic fragments. The sulfoxide derivative of both Tß4 and Tß10 and the acetylated derivatives at lysine residues of Tß4 were also characterized. Different proteoforms of prothymosin α, parathymosin α, thymosin α1 and thymosin α11 together with diverse proteolytic fragments were identified too. CONCLUSION: The clinical and prognostic significance and the origin of these proteoforms have to be deeply investigated.


Assuntos
Espectrometria de Massas/métodos , Timosina/análise , Adulto , Líquidos Corporais/química , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Recém-Nascido , Prognóstico , Precursores de Proteínas/análise , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Timalfasina , Timosina/análogos & derivados , Timosina/química , Timosina/metabolismo
6.
Mol Biosyst ; 11(6): 1668-83, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25909245

RESUMO

A top-down/bottom-up integrated proteomic approach based on LC-MS and 2-DE analysis was applied for comparative characterization of medulloblastoma and pilocytic astrocytoma posterior cranial fossa pediatric brain tumor tissues. Although rare, primary brain tumors are the most frequent solid tumors in the pediatric age. Among them the medulloblastoma is the prevalent malignant tumor in childhood while pilocytic astrocytoma is the most common, rarely showing a malignant progression. Due to the limited availability of this kind of sample, the study was applied to pooled tumor tissues for a preliminary investigation. The results showed different proteomic profiles of the two tumors and evidenced interesting differential expression of several proteins and peptides. Top-down proteomics of acid-soluble fractions of brain tumor homogenates ascribed a potential biomarker role of malignancy to ß- and α-thymosins and their truncated proteoforms and to C-terminal truncated (des-GG) ubiquitin, resulting exclusively detected or over-expressed in the highly malignant medulloblastoma. The bottom-up proteomics of the acid-soluble fraction identified several proteins, some of them in common with 2-DE analysis of acid-insoluble pellets. Peroxiredoxin-1, peptidyl-prolyl cis-trans isomerase A, triosephosphate isomerase, pyruvate kinase PKM, tubulin beta and alpha chains, heat shock protein HSP-90-beta and different histones characterized the medulloblastoma while the Ig kappa chain C region, serotransferrin, tubulin beta 2A chain and vimentin the pilocytic astrocytoma. The two proteomic strategies, with their pros and cons, well complemented each other in characterizing the proteome of brain tumor tissues and in disclosing potential disease biomarkers to be validated in a future study on individual samples of both tumor histotypes.


Assuntos
Astrocitoma/química , Neoplasias Encefálicas/química , Meduloblastoma/química , Proteoma/análise , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Criança , Pré-Escolar , Humanos , Meduloblastoma/metabolismo , Proteínas/análise , Proteínas/química , Proteínas/metabolismo , Proteoma/química , Proteoma/metabolismo , Proteômica
7.
J Proteome Res ; 13(11): 4594-606, 2014 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-25254300

RESUMO

Liquid chromatography in coupling with high-resolution ESI-LTQ-Orbitrap mass spectrometry was applied for a proteomic study of pediatric pilocytic astrocytoma brain tumor intracystic fluid by an integrated top-down/bottom-up platform. Both of the proteomic strategies resulted complementary and support each other in contributing to a wide characterization of the protein and peptide content of the tumor fluid. Top-down approach allowed to identify several proteins and peptides involved in different biological activities together with the characterization of interesting proteoforms such as fibrinopeptide A and its truncated form, fibrinopeptide B, complement C3f fragments, ß-thymosin peptides, ubiquitin, several apolipoproteins belonging to A and C families, apolipoprotein J and D, and cystatin C. Of particular interest resulted the identification of a N-terminal truncated cystatin C proteoform, likely involved in immune response mechanism modulations and the identification of oxidized and glycosylated apolipoproteins including disulfide bridge dimeric forms. The bottom-up approach confirmed some of the experimental data findings together with adding the characterization of high-molecular-mass proteins in the samples. These data could contribute to elucidate the molecular mechanisms involved in onset and progression of the disease and cyst development.


Assuntos
Astrocitoma/metabolismo , Líquido Cístico/metabolismo , Proteômica/métodos , Criança , Cromatografia Líquida de Alta Pressão , Cistatina C/metabolismo , Humanos , Espectrometria de Massas
8.
Electrophoresis ; 35(15): 2172-83, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24729313

RESUMO

The combination of top-down and bottom-up platforms was utilized for the LC-MS proteomic characterization of the intracystic fluid of adamantinomatous craniopharyngioma pediatric brain tumor disease. Proteins and peptides characterization was achieved by high-resolution LC-ESI-LTQ-Orbitrap-MS analysis while low-resolution LC-ESI-IT-MS was applied for the complete screening of the samples and the evaluation of the protein distribution within patients. Top-down analyses were applied to liquid/liquid extracted samples while bottom-up analyses were performed after trypsin digestion of both untreated and pretreated samples. The two proteomic approaches were complementary for the characterization of the proteome of craniopharyngioma intracystic fluid. Proteins and peptides involved in inflammation, mineralization processes and lipid transport were identified, in agreement with the calcium flecks, cholesterol granules and bone residues characteristic of this fluid. Apolipoprotein A-I, A-II, C-I and J, hemoglobin fragments, ubiquitin, α-2-HS-glycoprotein or fetuin A, α-1-antichymotrypsin, vitamin D binding protein, and α-1-acid glycoprotein were characterized. These data could be relevant for the comprehension of the processes involved in the pathogenesis of the disease and the development of the cyst and could contribute to the individuation of therapeutic targets for the reduction of the cyst volume delaying and/or avoiding invasive surgical treatments.


Assuntos
Craniofaringioma/química , Líquido Cístico/química , Neoplasias Hipofisárias/química , Proteoma/análise , Proteômica/métodos , Adolescente , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Espectrometria de Massas , Fragmentos de Peptídeos/análise , Tripsina
9.
Electrophoresis ; 34(18): 2674-82, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23857244

RESUMO

Thymosin ß4 (Tß4) is a peptide present in almost any tissue and in extracellular media in mammals, having multiple amazing functions as wound healing, stimulation of angiogenesis, and suppression of inflammation. This study describes its determination in saliva through CE-MS using multiple ions monitoring scan mode by isolating the four most intense multicharged ions present in the MS spectra of the peptide. This scan modality, by reducing the baseline noise and interferences, increases the sensitivity and specificity in biological matrices. The CE-MS separation was optimized by studying different parameters influencing CE analysis, sample injection, and MS ionization, that is, the nebulizer gas flow, the sheath liquid, and BGE composition. The proposed technique can unambiguously identify in short time Tß4 in saliva after a very fast and reduced sample pretreatment procedure. The method was validated for quantitation showing linearity of the response in the range 0.25 (lower limit of quantification) to 4 µM (average R2 0.996 ± 0.005) and intra- and interassay precision and accuracy at three different concentrations with RSD values in the range of 7­16%. It was successfully applied to the analysis of Tß4 in whole saliva showing a variable peptide content from individual to individual (in the range of 0.3­1.4 µM) and in different days from the same individual. CE-MS in multiple ions monitoring scan mode provides a fast, selective, and economic method requiring only very few microliters of sample.


Assuntos
Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Saliva/química , Timosina/análise , Adulto , Idoso , Sequência de Aminoácidos , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
10.
Dent Mater ; 29(8): e153-60, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23764026

RESUMO

OBJECTIVE: Various protective effects of N-acetylcysteine (NAC) against triethylene glycol dimethacrylate (TEGDMA)-induced cell damage have been demonstrated, but so far there is no evidence on NAC direct monomer detoxification mechanism. Here, we hypothesized that NAC might reduce TEGDMA cytotoxicity due to direct NAC adduct formation. METHODS: We measured the cytotoxic effects of TEGDMA in presence and in absence of NAC by MTT test. Then we analyzed the presence of TEGDMA-NAC adduct formation in extracellular and intracellular compartments by capillary electrophoresis-UV detection (CE-UV) and capillary electrophoresis-mass spectrometry (CE-MS) analytical techniques. Moreover, we quantified the effective intracellular and extracellular TEGDMA concentrations through HPLC in the presence and absence of 10 mmol/L NAC. RESULTS: TEGDMA reduced 3T3 cell vitality in a dose- and time-dependent manner, while NAC decreased monomer cytotoxicity and extracellular monomer concentrations by a direct reaction with TEGDMA. The adducts between the two molecules were detected both in the presence and absence of cell. Moreover a signal ascribed to the methacrylic acid was present in the CE-UV electropherogram of cellular lysates obtained after incubation with TEGDMA. SIGNIFICANCE: Our results suggest that in vitro detoxification capability of NAC against TEGDMA-induced cell damage might occur also through the formation of NAC-TEGDMA adduct.


Assuntos
Acetilcisteína/farmacologia , Fibroblastos/efeitos dos fármacos , Polietilenoglicóis/toxicidade , Ácidos Polimetacrílicos/toxicidade , Substâncias Protetoras/farmacologia , Acetilcisteína/química , Animais , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão/métodos , Corantes , Citosol/química , Relação Dose-Resposta a Droga , Interações Medicamentosas , Eletroforese Capilar , Espaço Extracelular/química , Metacrilatos/análise , Camundongos , Polietilenoglicóis/análise , Polietilenoglicóis/química , Ácidos Polimetacrílicos/análise , Ácidos Polimetacrílicos/química , Substâncias Protetoras/química , Células Swiss 3T3 , Espectrometria de Massas em Tandem/métodos , Sais de Tetrazólio , Tiazóis , Fatores de Tempo , Raios Ultravioleta
11.
Childs Nerv Syst ; 29(6): 951-60, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23503632

RESUMO

BACKGROUND: Adamantinomatous craniopharyngioma is the third most recurrent paediatric brain tumour. Although histologically benign, it behaves aggressively as a malignant tumour due to invasion of the hypothalamus and visual pathways. Surgery is still the first and almost the only mode of treatment, although serious damage can occur as a consequence of tumour localization. The proteomic characterization of the intracystic tumoural fluid could contribute to the comprehension of the tumorigenesis processes and to the development of therapeutic targets to reduce cyst volume, allowing less invasive surgery and/or delay of the radical resection of the tumour mass and the collateral serious effects. METHODS: Intracystic fluid was analysed by a LC-ESI-IT-MS top-down platform after acidification, deproteinization and chloroform liquid/liquid extraction. FINDINGS: Thymosin ß4 and ß10 peptides were for the first time identified in the intracystic fluid of adamantinomatous craniopharyngioma by low- and high-resolution MS analysis coupled with LC. The two peptides showed the same distribution trend in the analysed samples. Thymosin ß4 and ß10 were present in 77 % of the analysed samples. These peptides were not found in the cerebrospinal fluid available for two patients. INTERPRETATION: The presence of ß-thymosins in the intracystic fluid of the tumour confirmed the secretion of these proteins in the extracellular environment. Due to their G-actin-sequestering activity and antiapoptotic and anti-inflammatory properties, these peptides could be strictly involved in both tumour progression and cyst development and growth.


Assuntos
Craniofaringioma/líquido cefalorraquidiano , Craniofaringioma/metabolismo , Neoplasias Hipofisárias/líquido cefalorraquidiano , Neoplasias Hipofisárias/metabolismo , Timosina/líquido cefalorraquidiano , Adolescente , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Craniofaringioma/cirurgia , Endoscopia , Feminino , Humanos , Masculino , Espectrometria de Massas , Complexos Multiproteicos , Neoplasias Hipofisárias/cirurgia
12.
J Chromatogr A ; 1267: 170-7, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-22841664

RESUMO

A CE-tandem MS method was optimised and validated for selective and specific determination of LVV- and VV-hemorphin-7 peptides in cerebrospinal fluid. These two small peptides originate from haemoglobin beta chains. They possess relevant biological activity and recently a potential biomarker role in posterior cranial fossa paediatric brain tumour disease was evidenced. The separation was optimised using formic acid as background electrolyte and a water/methanol mixture, containing 0.1% (v/v) formic acid, as sheath liquid. The two peptides, differing in only one amino acid of the sequence at the N-terminal side were baseline separated in less than 15 min. The method allowed a very reduced and rapid sample pretreatment and was successfully applied to hemorphins determination in patient samples without matrix interferences. The method successfully passed bioanalytical validation showing linearity, accuracy and precision data on cerebrospinal fluid matrix within the acceptable values. The analysis of cerebrospinal fluid of patients affected by different posterior cranial fossa tumour forms confirmed our previous findings showing the absence of hemorphins in the pre-surgical cerebrospinal fluid and their presence in the post-ones and controls. The present method saves costs and time due to capillary electrophoresis miniaturisation and to the absence of chromatographic column and gradient elution and allows numerous injections per sample starting from few microlitres of cerebrospinal fluid.


Assuntos
Neoplasias Encefálicas/líquido cefalorraquidiano , Eletroforese Capilar/métodos , Hemoglobinas/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidiano , Espectrometria de Massas em Tandem/métodos , Neoplasias Encefálicas/diagnóstico , Criança , Pré-Escolar , Feminino , Humanos , Masculino
13.
Proteomics ; 12(13): 2158-66, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22623401

RESUMO

Posterior cranial fossa is the most frequent location of pediatric brain tumors. Its diagnosis is currently performed by postsurgery histopathology and the identification of biomarkers in cerebrospinal fluid (CSF) could provide a less invasive tool. Patient CSF was collected during surgery before the tumor removal (PRE-CSF) and 6 days after the resection (POST-CSF) and analyzed by top down LC-MS proteomics for comparison. The PRE-CSFs generally exhibited a less complex LC-MS profile than the relative POST-CSFs suggesting a suppressive role of the tumor toward proteins and peptides production or release. Particularly, a panel of peptides, identified as alpha- and beta-hemoglobin chains fragments, were generally absent in the PRE-CSF and present in the POST ones independently from contaminant blood hemoglobin. Among them, the LVV- and VV-hemorphin-7 showed the most repeatable trend and with a few remarkable exceptions: their unusual absence in POST surgery CSF was in fact interestingly correlated to the presence of tumor in the patient despite surgery due to metastases or to subtotal resection. These results ascribed a relevant biological role to LVV- and VV-h7 peptides in the disease and a strong potential as biomarkers. Their analysis in POST surgery CSF could be used to predict patient prognosis.


Assuntos
Biomarcadores Tumorais/líquido cefalorraquidiano , Neoplasias Encefálicas/líquido cefalorraquidiano , Hemoglobinas/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteômica/métodos , Adolescente , Sequência de Aminoácidos , Biomarcadores Tumorais/química , Neoplasias Encefálicas/patologia , Criança , Pré-Escolar , Fossa Craniana Posterior/patologia , Feminino , Hemoglobinas/química , Humanos , Lactente , Recém-Nascido , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química
14.
J Pharm Biomed Anal ; 53(5): 1161-9, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20674216

RESUMO

The increasing attention now paid to the elucidation of human proteome strengthened the development of analytical instruments able to provide reliable proteins and peptides quantitation and characterization in biological fluids and tissues. Emerging from proteomics, clinical proteomics exclusively considers its biomedical applications. It evaluates, often by high-throughput comparative platforms, the protein and peptide variations in body fluids, cells and tissues under different physiological and pathological conditions with the aim of discovering disease biomarkers. Among the available analytical methodologies, mass spectrometry in coupling with liquid chromatography or capillary electrophoresis demonstrated to be the eligible technique for protein detection and identification. This review summarizes the most recent applications of capillary electrophoresis-mass spectrometry to clinical proteomics, focusing on capillary zone electrophoresis separation mode and ESI and MALDI ionizations, which are the most frequently applied capillary electrophoresis-mass spectrometry hyphenated techniques.


Assuntos
Espectrometria de Massas/tendências , Proteômica/tendências , Animais , Biomarcadores/análise , Eletroforese Capilar/tendências , Humanos , Nefropatias/sangue , Nefropatias/diagnóstico , Nefropatias/urina
15.
J Sep Sci ; 33(16): 2385-93, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20535752

RESUMO

In this review, the recent contribution of CE-MS technology to the analysis of amino acids, as well as the advantages of the hyphenation and the technologies involved in the instrumental coupling are reported. Different sections are dedicated to the recent contributions of CE-MS to the analysis of protein amino acids and their post-translational modifications, such as phosphorylation and sulfation. CE-MS analysis of some amino acid derivatives, such as the free methylated-derivatives of arginine is also discussed. A section is specifically devoted to the CE-MS applications in the field of chiral separation of D- and L-amino acid enantiomers.


Assuntos
Aminoácidos/análise , Eletroforese Capilar , Espectrometria de Massas
16.
Electrophoresis ; 31(11): 1894-902, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20432477

RESUMO

A CE ion trap tandem MS method was optimised for the analysis of arginine, monomethyl- and (symmetric and asymmetric) dimethylarginines in human plasma after a very reduced sample pretreatment step involving a simple protein precipitation with ACN. Several parameters affecting the analytes MS ionization and the capillary electrophoretic separation were carefully studied and optimised. The complete separation of arginine, monomethylarginine and symmetric and asymmetric dimethylarginine was obtained in formic acid BGE in short analysis time with high specificity due to MS(2) detection of specific analytes fragments. In order to achieve the detection sensitivity suitable for the analysis of asymmetric and symmetric dimethylarginine in human plasma, the field amplified sample injection was applied. Due to stacking effects, this methodology allowed to operate a consistent on-line preconcentration of the analytes before running the electrophoresis. The method was validated for linearity, repeatability, recovery and accuracy and applied to the quantitative analysis of arginine, monomethyl- and dimethylarginines in human plasma of healthy subjects.


Assuntos
Acetonitrilos/sangue , Arginina/análogos & derivados , Arginina/sangue , Eletroforese Capilar/métodos , Análise de Injeção de Fluxo/métodos , Espectrometria de Massas em Tandem/métodos , ômega-N-Metilarginina/sangue , Acetonitrilos/química , Arginina/química , Humanos , Modelos Lineares , Metilação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , ômega-N-Metilarginina/química
17.
Biomaterials ; 31(9): 2508-16, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20022629

RESUMO

Cytotoxicity of the dental resin monomer 2-hydroxyethyl methacrylate (HEMA) and the protective effects of N-acetyl cysteine (NAC) on monomer-induced cell damage are well demonstrated. The aim of our study was to analyze the hypothesis that the protection of NAC from HEMA cytotoxicity might be due to direct NAC adduct formation. To this end, using HPLC we first measured the actual intracellular HEMA concentrations able to cause toxic effects on 3T3-fibroblasts and then determined the decrease in intracellular and extracellular HEMA levels in the presence of NAC. In addition, by capillary electrophoresis coupled with mass spectrometry analysis (CE-MS), we evaluated NAC-HEMA adduct formation. HEMA reduced 3T3 cell vitality in a dose- and time-dependent manner. The concentration of HEMA inside the cells was 15-20 times lower than that added to the culture medium for cell treatment (0-8 mmol/L). In the presence of 10 mmol/L NAC, both intracellular and extracellular HEMA concentrations greatly decreased in conjunction with cytotoxicity. NAC-HEMA adducts were detected both in the presence and absence of cells. Our findings suggest that the in vitro detoxification ability of NAC against HEMA-induced cell damage occurs through NAC adduct formation. Moreover, we provide evidence that the actual intracellular concentration of HEMA able to cause cytotoxic effects is at least one magnitude lower than that applied extracellularly.


Assuntos
Acetilcisteína/farmacologia , Metacrilatos/química , Metacrilatos/toxicidade , Células 3T3 , Acetilcisteína/química , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Espectrometria de Massas , Camundongos
18.
Artigo em Inglês | MEDLINE | ID: mdl-18656427

RESUMO

Capillary electrophoresis (CE) coupled to tandem mass spectrometry was applied to the chiral separation of baclofen using sulfobutylether-beta-cyclodextrin chiral selector in partial filling counter current mode. On-line UV detection was simultaneously used. Method optimization was performed by studying the effect of cyclodextrin and BGE concentration as well as sheath liquid composition on analyte migration time and enantiomeric resolution. The cyclodextrin showed stereoselective complexation towards baclofen enantiomers, allowing chiral resolution at low concentration. The CE capillary protrusion from the ESI needle relevantly affected the chiral resolution and the analyte migration time. Complete enantiomeric separation was obtained by using 0.25 M formic acid BGE containing 1.75 mM of chiral selector and water/methanol (30:70, v/v) 3% formic acid as sheath liquid. The method exhibited a LOD of 0.1 microg/mL (racemic concentration) in MS3 product ion scan mode of detection and was applied to the analysis of racemic baclofen in pharmaceutical formulations.


Assuntos
Baclofeno/isolamento & purificação , Eletroforese Capilar/métodos , Espectrometria de Massas em Tandem/métodos , Eletroforese Capilar/instrumentação , Formiatos/química , Preparações Farmacêuticas/química , Sensibilidade e Especificidade , Estereoisomerismo , beta-Ciclodextrinas
19.
Anal Bioanal Chem ; 390(6): 1637-44, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18264699

RESUMO

A capillary electrophoresis (CE) method coupled to electrospray ionization ion trap tandem mass spectrometry (ESI-IT-MS/MS) is described for the rapid analysis of carnitine, acetylcarnitine, and propionylcarnitine in human plasma. Optimization of the procedure was achieved by a reduced sample pretreatment and after examining several physicochemical parameters that influence both the CE separation and the MS analytes detection. The analysis of total carnitine in human plasma after hydrolysis of short-chain metabolites is also shown. The analysis of carnitine and metabolites was obtained in less than 10 min using a 200 mM ammonium formate buffer, pH 2.5, with high sensitivity and specificity using the MS detection in product ion scan mode. The method was tested for quantitative recovery using dialyzed human plasma as matrix and showed linearity in the concentrations ranges 20-160, 1-32, and 0.25-8 microM for carnitine, acetylcarnitine, and propionylcarnitine with (squared) correlation coefficients of 0.9984, 0.9995, and 0.9991, respectively. The intraday and intermediate analysis repeatability and accuracy are within 15% of relative standard deviation (RSD) at low, medium, and high concentration and within/or slight exceeding 20% at the lower limit of quantitation (LLOQ). The method is sensitive for determining carnitine and its metabolites in human plasma with high specificity.


Assuntos
Carnitina/sangue , Carnitina/química , Eletroforese Capilar/métodos , Espectrometria de Massas em Tandem/métodos , Humanos , Estrutura Molecular , Fatores de Tempo
20.
J Chromatogr A ; 1150(1-2): 320-6, 2007 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-17028001

RESUMO

A capillary electrophoresis apparatus was used as sampler for flow injection analysis (FIA) in tandem mass spectrometry of L-carnitine and its acetyl- and propionyl-metabolites in human plasma. The capillary electrophoresis instrument was coupled to the ion trap mass spectrometer by an electrospray ionization coaxial sheath liquid interface. The electrophoresis capillary introduced the sample directly into the source by applying a prolonged sample injection. The use of the capillary electrophoresis apparatus miniaturised the FIA procedure, substantially reducing the quantities of solvents and samples used, and allowed rapid automated sequential analyses. The method was optimised and validated using a dialyzed human plasma matrix. The plasma samples were analysed after a simple, rapid deproteinisation procedure with acetonitrile and diluted 70 times before direct injection into the mass spectrometer for product ion scan MS/MS analysis in positive ionisation. The total analysis time was 5 min, including capillary preconditioning and acquisition time (3 min). The method was sensitive, allowing the determination of L-, L-acetyl- and L-propionyl-carnitines at 140, 14 and 3.6 nM concentrations (injected values) corresponding to lower limit of quantitation values in plasma of 10, 1 and 0.25 microM, respectively. The method was processed for full validation and applied to the analysis of L-carnitine and its short chain derivatives in human plasma samples.


Assuntos
Carnitina/sangue , Eletroforese Capilar/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Carnitina/química , Humanos , Estrutura Molecular , Reprodutibilidade dos Testes
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