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Optimized sample preparation of endoscopic collected pancreatic fluid for SDS-PAGE analysis

Authors

  • Joao A. Paulo,

    1. Department of Pathology, Children's Hospital Boston and Harvard Medical School, Boston, MA, USA
    2. Proteomics Center at Children's Hospital Boston, Boston, MA, USA
    3. Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, MA, USA
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  • Linda S. Lee,

    1. Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, MA, USA
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  • Bechien Wu,

    1. Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, MA, USA
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  • Kathryn Repas,

    1. Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, MA, USA
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  • Peter A. Banks,

    1. Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, MA, USA
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  • Darwin L. Conwell,

    1. Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, MA, USA
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  • Hanno Steen

    Corresponding author
    1. Department of Pathology, Children's Hospital Boston and Harvard Medical School, Boston, MA, USA
    2. Proteomics Center at Children's Hospital Boston, Boston, MA, USA
    • Children's Hospital Boston, Department of Pathology/Enders 1130, 320 Longwood Ave, Boston, MA 02115, USA Fax: +1-617-730-0168
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Abstract

The standardization of methods for human body fluid protein isolation is a critical initial step for proteomic analyses aimed to discover clinically relevant biomarkers. Several caveats have hindered pancreatic fluid proteomics, including the heterogeneity of samples and protein degradation. We aim to optimize sample handling of pancreatic fluid that has been collected using a safe and effective endoscopic collection method (endoscopic pancreatic function test). Using SDS-PAGE protein profiling, we investigate (i) precipitation techniques to maximize protein extraction, (ii) auto-digestion of pancreatic fluid following prolonged exposure to a range of temperatures, (iii) effects of multiple freeze–thaw cycles on protein stability, and (iv) the utility of protease inhibitors. Our experiments revealed that TCA precipitation resulted in the most efficient extraction of protein from pancreatic fluid of the eight methods we investigated. In addition, our data reveal that although auto-digestion of proteins is prevalent at 23 and 37°C, incubation on ice significantly slows such degradation. Similarly, when the sample is maintained on ice, proteolysis is minimal during multiple freeze–thaw cycles. We have also determined the addition of protease inhibitors to be assay-dependent. Our optimized sample preparation strategy can be applied to future proteomic analyses of pancreatic fluid.

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