1. High reproducibility of two-dimensional liquid chromatography using pH-driven fractionation with a pressure-resistant electrode
- Author
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Daniel Gonzalez-Dunia, Frédéric Pont, Elsa Suberbielle, Centre de Physiopathologie Toulouse Purpan (CPTP), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Plateau Technique de Spectrométrie de Masse, Institut Claude de Préval (ICP), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Toulouse [Toulouse]-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Toulouse [Toulouse]-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées
- Subjects
Proteomics ,Two-dimensional liquid chromatography ,MESH: Hydrogen-Ion Concentration ,Non-porous silica ,Chromatogram comparison ,Clinical Biochemistry ,Analytical chemistry ,MESH: Vero Cells ,Fractionation ,01 natural sciences ,Biochemistry ,pH meter ,High-performance liquid chromatography ,Analytical Chemistry ,03 medical and health sciences ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Chlorocebus aethiops ,Animals ,Sample preparation ,Peak concordance ,MESH: Animals ,Vero Cells ,030304 developmental biology ,0303 health sciences ,Reproducibility ,Chromatography ,Chemistry ,MESH: Proteomics ,010401 analytical chemistry ,PF2D ,Reverse phase high-performance liquid chromatography ,Reproducibility of Results ,Cell Biology ,General Medicine ,Repeatability ,Reversed-phase chromatography ,Hydrogen-Ion Concentration ,MESH: Cercopithecus aethiops ,0104 chemical sciences ,MESH: Reproducibility of Results ,Two-dimensional chromatography ,HPLC ,MESH: Chromatography, Liquid ,Chromatofocusing ,Chromatography, Liquid - Abstract
International audience; Automated two-dimensional liquid chromatography using the PF2D system from Beckman Coulter provides a fractionation platform well suited for differential proteomic studies. To date, the reliability and reproducibility of PF2D has not been accurately tested. Here, we used an optimized software and a pressure-resistant pH electrode, allowing a precise and reproducible control of the pH limits for each fraction during PF2D. We tested the reliability of this improved system by performing several rounds of fractionation using the same protein extract. Three UV maps were generated, leading to 54 chromatograms and more than 3000 protein peaks. Using semi-automated software for peak-to-peak comparison between 2D-LC chromatograms, we demonstrate that the peak concordance is very high. The rates of concordance were higher in the second dimension repeatability tests, indicating that the limiting factors for 2D-LC reproducibility rely on the pI fractionation and sample preparation steps. The reproducibility between maps was closely related to pH curves similarities, further stressing the need of careful pH adjustment and precise electrode calibration.
- Published
- 2008
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