Abstract
We have carried out a comprehensive characterization of human bile to define the bile proteome. Our approach involved fractionation of bile by one-dimensional gel electrophoresis and lectin affinity chromatography followed by liquid chromatography tandem mass spectrometry. Overall, we identified 87 unique proteins, including several novel proteins as well as known proteins whose functions are unknown. A large majority of the identified proteins have not been previously described in bile. Using lectin affinity chromatography and enzymatically labeling of asparagine residues carrying glycan moieties by (18)O, we have identified a total of 33 glycosylation sites. The strategy described in this study should be generally applicable for a detailed proteomic analysis of most body fluids. In combination with "tagging" approaches for differential proteomics, our method could be used for identification of cancer biomarkers from any body fluid.
Highlights
We have carried out a comprehensive characterization of human bile to define the bile proteome
We provide definitive evidence for a large number of N-linked glycosylation sites on these proteins using lectin affinity chromatography followed by 18O-labeling of the glycan attachment site by peptide N-glycosidase F (PNGaseF) treatment
Sample Preparation and Fractionation—Our initial strategy was to identify protein components in human bile by onedimensional SDS-PAGE of crude bile followed by in-gel trypsin digestion and subsequent identification of the proteins by LC-MS/MS
Summary
We have carried out a comprehensive characterization of human bile to define the bile proteome. Biliary tract cancers are notoriously challenging to diagnose and treat At present, only those patients with a completely resectable cancer achieve a modest 5-year survival. Changes that occur during the transformation of a healthy cell into a neoplastic cell can result in protein alterations including changes in abundance, protein modification, enzymatic activity, or subcellular localization Identifying and understanding these changes is an underlying theme in cancer proteomics [9, 10]. We provide the first comprehensive proteomic study of human bile fluid using a liquid chromatography and tandem mass spectrometric (LC-MS/MS) approach. We have elucidated the proteome of bile fluid using multiple fractionation techniques and affinity enrichment methods to identify several proteins that have not been previously described in bile. We provide definitive evidence for a large number of N-linked glycosylation sites on these proteins using lectin affinity chromatography followed by 18O-labeling of the glycan attachment site by peptide N-glycosidase F (PNGaseF) treatment
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