TY - JOUR
T1 - Proteome profile of human urine with two-dimensional liquid phase fractionation
AU - Soldi, Monica
AU - Sarto, Cecilia
AU - Valsecchi, Cristina
AU - Magni, Fulvio
AU - Proserpio, Vanessa
AU - Ticozzi, Davide
AU - Mocarelli, Paolo
PY - 2005/7
Y1 - 2005/7
N2 - Two-dimensional liquid chromatography separation (2-DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two-dimensional gel electrophoresis (2-DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0-8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix-assisted laser desorption/ionization-time of flight/mass spectrometry analysis for identification. The results showed that the 2-DL provides high reproducibility of two-dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2-DE gels. In addition, this system also allows to separate particularly proteins with 40-9 kDa molecular weight.
AB - Two-dimensional liquid chromatography separation (2-DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two-dimensional gel electrophoresis (2-DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0-8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix-assisted laser desorption/ionization-time of flight/mass spectrometry analysis for identification. The results showed that the 2-DL provides high reproducibility of two-dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2-DE gels. In addition, this system also allows to separate particularly proteins with 40-9 kDa molecular weight.
KW - Body fluids
KW - Two-dimensional liquid chromatography
KW - Urinary protein
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U2 - 10.1002/pmic.200401269
DO - 10.1002/pmic.200401269
M3 - Article
C2 - 15912557
AN - SCOPUS:22044442841
VL - 5
SP - 2641
EP - 2647
JO - Proteomics
JF - Proteomics
SN - 1615-9853
IS - 10
ER -