Optimized preparation of urine samples for two-dimensional electrophoresis and initial application to patient samples

Lafitte, Daniel; Dussol, Bertrand; Andersen, Sebastian; Vazi, Alain; Dupuy, P.; Jensen, Ole N.; Berland, Yvon; Verdier, Jean‐Michel

doi:10.1016/s0009-9120(02)00362-4

Cited by 72 publications

(60 citation statements)

References 28 publications

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“…The aggregate concentration in plasma of globulins with a molecular size similar to albumin, proteins such as transferrin, ␣ 1 -acid glycoprotein, ␣ 1 -proteinase inhibitor, ␣ 1 -antichymotrypsin, ␣ 2 -HS glycoprotein, hemopexin, Gc-globulin, and many others (31 ), in healthy individuals is ϳ25% that of albumin; this aggregate concentration is even higher during acutephase reactions (32 ). Because the passage of plasma proteins through the glomerular barrier into urine is primarily a size-dependent process with a size-exclusion limit approximately the size of albumin (33 ), it is not surprising that all of the albumin-sized plasma components are also found in urine (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(33)(34)(35)(36)(37)(38)(39). There is considerable variation in the reported proportions of individual globulins relative to albumin in urine (Table 3).…”

Section: Discussionmentioning

confidence: 99%

“…Studies of reference intervals for albumin values from the HPLC assay indicate that ranges are ϳ2-fold higher than for immunoassays (11 ); therefore, efforts to apply the same diagnostic cutoff values that have been established for immunoassay methods to albumin measurements by size-exclusion HPLC, as in some recent studies (6 -9 ), are inappropriate. From the standpoint of interest in proteomic analysis of urine (15)(16)(17), size-exclusion chromatography appears to be a useful technique for producing a low-resolution size fractionation of urinary proteins. This fractionation appears to be a useful preparative step and precursor to other analytical techniques, some of which were applied to specific chromatographic fractions in this study.…”

Section: Discussionmentioning

confidence: 99%

“…Although albumin is the most abundant plasma protein, there are many other plasma proteins of approximately the same molecular size, such as transferrin, ␣ 1 -proteinase inhibitor, hemopexin, ␣ 1 -acid glycoprotein, ␣ 1 -antichymotrypsin, Gc-globulin, ␣ 2 -HS glycoprotein, and transthyretin, that are major urinary components (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Fundamental principles suggest that size-exclusion HPLC will not resolve albumin from other urinary proteins of similar molecular size.…”

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confidence: 99%

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Coelution of Other Proteins with Albumin during Size-Exclusion HPLC: Implications for Analysis of Urinary Albumin

et al. 2006

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Background: Size-exclusion HPLC has been used as an alternative to immunoassays for quantifying urinary albumin (microalbumin). Systematically higher values for the HPLC method have been proposed to result from nonimmunoreactive albumin. Methods: We evaluated separation of purified proteins and urinary components by size-exclusion HPLC using a Zorbax Bio Series GF-250 column eluted with phosphate-buffered saline. Urinary components eluting in the "albumin" peak were analyzed by mass spectrometry and reversed-phase HPLC. Results: Several proteins, such as transferrin, ␣ 1 -proteinase inhibitor, ␣ 1 -acid glycoprotein, and ␣ 2 -HS glycoprotein, analyzed as purified components, were not resolved from albumin by size-exclusion HPLC. Peaks for other proteins, such as IgG and urinary components identified as dimers of ␣ 1 -microglobulin and immunoglobulin light chains, overlapped with the albumin peak. Profiles of urine specimens showed variable amounts of components overlapping with albumin. Furthermore, the albumin peak obtained by size-exclusion HPLC was found by mass spectrometry and reversedphase HPLC to contain multiple components in addition to albumin. Conclusions: Size-exclusion HPLC does not resolve albumin from several other proteins in urine. The albumin peak resolved by this technique, although predominantly composed of albumin, contains several coelut-

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Coelution of Other Proteins with Albumin during Size-Exclusion HPLC: Implications for Analysis of Urinary Albumin

et al. 2006

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“…Optimization of sample preparation is a necessary first step for urinary proteome analysis and sample desalting using SPE, precipitation or dialysis is often required [265][266][267][268][269]. Acetone appears to precipitate more acidic and hydrophilic proteins, whereas ultracentrifugation fractionates more basic, hydrophobic, and membrane proteins [266].…”

Section: Analysis and Characterization Of Urine Proteomementioning

confidence: 99%

Human body fluid proteome analysis

2006

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The focus of this article is to review the recent advances in proteome analysis of human body fluids, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, and amniotic fluid, as well as its applications to human disease biomarker discovery. We aim to summarize the proteomics technologies currently used for global identification and quantification of body fluid proteins, and elaborate the putative biomarkers discovered for a variety of human diseases through human body fluid proteome (HBFP) analysis. Some critical concerns and perspectives in this emerging field are also discussed. With the advances made in proteomics technologies, the impact of HBFP analysis in the search for clinically relevant disease biomarkers would be realized in the future.

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“…20,51 As proteínas solúveis da urina são derivadas em grande parte da filtração glomerular, a qual retarda a passagem de proteínas de elevado peso molecular. …”

Section: Análise Proteômica Aplicada Ao Câncer De Bexigaunclassified

Avaliação analítica de potenciais biomarcadores para câncer de bexiga em urina

Alberice¹

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A Deus pela dádiva da vida e força para vencer os obstáculos e alcançar meus objetivos.Aos meus pais Claudir e Aparecida pela oportunidade de estudar, por todo apoio e amor incondicional. Ao meu irmão Wellington pelo carinho e paciência.Ao meu namorado Camilo pelo companheirismo, amor, paciência e por me ajudar a ser uma pessoa melhor.Aos meus familiares que contribuíram e contribuem para o meu crescimento agradeço pelo apoio, carinho e torcida.Às minhas queridas amigas de infância Aline, Carol, Rachel, Giovana e Vanessa com as quais sei que posso contar independente da distância. Em especial agradeço à Rachel por me escutar e me entender, ainda que nenhuma palavra seja dita.Agradeço imensamente ao Prof. Dr. Emanuel, por todos esses anos de orientação e amizade, pela oportunidade e confiança no desenvolvimento do trabalho. Sou muito grata e honrada de poder ter crescido profissionalmente dentro do BioMicS. ResumoO câncer de bexiga é uma neoplasia urogenital que acomete homens e mulheres, sendo que somente no Brasil 8.600 novos casos ao ano são diagnosticados. Cistoscopia transuretral é a conduta padrão no diagnóstico e acompanhamento do câncer de bexiga. Entretanto, tal procedimento é extremamente invasivo e doloroso além de ter elevado custo e não garantir todos os resultados. Assim, busca-se por marcadores moleculares que possam auxiliar no diagnóstico e progressão do câncer de bexiga, bem como diminuir a necessidade de exames invasivos no acompanhamento de pacientes tratados. Nesse sentido, a urina tem papel de destaque como fonte de biomarcadores devido principalmente ao seu caráter não invasivo.Nesse trabalho foram utilizadas duas abordagens 'ômicas': proteômica e metabolômica, para a busca de biomarcadores em urina para o diagnóstico e prognóstico do câncer de bexiga, respectivamente. Com a abordagem proteômica buscou-se apenas por biomarcadores para o diagnóstico da doença e, utilizando as técnicas de eletroforese 2-DE, OFFGEL e MS, juntamente com análise estatística multivariada, foi possível identificar 32 proteínas que apresentam-se como potenciais marcadores para o câncer de bexiga. A abordagem metabolômica foi empregada para a busca de biomarcadores para reincidência e progressão da doença. As técnicas analíticas utilizadas nessa abordagem, LC-MS e CE-MS, mostraram-se complementares e, dos resultados obtidos com ambas e avaliados com análise estatística multivariada foi possível indicar 19 metabólitos para reincidência e 23 metabólitos para progressão do câncer de bexiga.Assim, neste trabalho explorou-se como as ciências 'ômicas', a qual abrange técnicas analíticas de high-throughput, estatística multivariada e ferramentas de bioinformática auxiliando na descoberta de potenciais biomarcadores não invasivos para o diagnóstico e prognóstico do câncer de bexiga. Portanto, o presente estudo foi de grande importância e relevância à medida que ilustrou como tais técnicas podem potencialmente auxiliar no diagnóstico e prognóstico de doenças e contribuir para tratamentos personalizados no futuro, indicando ...

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Optimized preparation of urine samples for two-dimensional electrophoresis and initial application to patient samples

Cited by 72 publications

References 28 publications

Coelution of Other Proteins with Albumin during Size-Exclusion HPLC: Implications for Analysis of Urinary Albumin

Coelution of Other Proteins with Albumin during Size-Exclusion HPLC: Implications for Analysis of Urinary Albumin

Human body fluid proteome analysis

Avaliação analítica de potenciais biomarcadores para câncer de bexiga em urina

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