title: One Sample, One Shot - Evaluation of sample preparation protocols for the mass spectrometric proteome analysis of human bile fluid without extensive fractionation.
abstract: The proteome analysis of bile fluid represents a promising strategy to identify biomarker candidates for various diseases of the hepatobiliary system. However, to obtain substantive results in biomarker discovery studies large patient cohorts necessarily need to be analyzed. Consequently, this would lead to an unmanageable number of samples to be analyzed if sample preparation protocols with extensive fractionation methods are applied. Hence, the performance of simple workflows allowing for "one sample, one shot" experiments have been evaluated in this study. In detail, sixteen different protocols implying modifications at the stages of desalting, delipidation, deglycosylation and tryptic digestion have been examined. Each method has been individually evaluated regarding various performance criteria and comparative analyses have been conducted to uncover possible complementarities. Here, the best performance in terms of proteome coverage has been assessed for a combination of acetone precipitation with in-gel digestion. Finally, a mapping of all obtained protein identifications with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) revealed several proteins easily detectable in bile fluid. These results can build the basis for future studies with large and well-defined patient cohorts in a more disease-related context.
  
  
  
  Human bile fluid is a proximal body fluid and supposed to be a potential source of disease markers. However, due to its biochemical composition, the proteome analysis of bile fluid still represents a challenging task and is therefore mostly conducted using extensive fractionation procedures. This in turn leads to a high number of mass spectrometric measurements for one biological sample. Considering the fact that in order to overcome the biological variability a high number of biological samples needs to be analyzed in biomarker discovery studies, this leads to the dilemma of an unmanageable number of necessary MS-based analyses. Hence, easy sample preparation protocols are demanded representing a compromise between proteome coverage and simplicity. In the presented study, such protocols have been evaluated regarding various technical criteria (e.g. identification rates, missed cleavages, chromatographic separation) uncovering the strengths and weaknesses of various methods. Furthermore, a cumulative bile proteome list has been generated that extends the current bile proteome catalog by 248 proteins. Finally, a mapping with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) derived from tissue-based studies, revealed several of these proteins being easily and reproducibly detectable in human bile. Therefore, the presented technical work represents a solid base for future disease-related studies.
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum, Germany. Electronic address: dominik.megger@rub.de.
authors.forename: Dominik A
authors.initials: DA
authors.lastname: Megger
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum, Germany.
authors.forename: Juliet
authors.initials: J
authors.lastname: Padden
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Rosowski
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Uszkoreit
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Bracht
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Eisenacher
authors.affiliation: Department of Internal Medicine, Evangelisches Krankenhaus, Düsseldorf, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Gerges
authors.affiliation: Department of Internal Medicine, Evangelisches Krankenhaus, Düsseldorf, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: Neuhaus
authors.affiliation: Klinik für Innere Medizin und Gastroenterologie, Elisabeth Krankenhaus Essen, Essen, Germany.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Schumacher
authors.affiliation: Department of Internal Medicine, Evangelisches Krankenhaus Duisburg-Nord, Duisburg, Germany.
authors.forename: Jörg F
authors.initials: JF
authors.lastname: Schlaak
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Sitek
mesh_terms: D001646:Bile / Q000737:chemistry*; D001650:Bile Duct Neoplasms / Q000737:chemistry / Q000175:diagnosis; D014408:Biomarkers, Tumor / Q000032:analysis; D006528:Carcinoma, Hepatocellular / Q000737:chemistry / Q000175:diagnosis; D005591:Chemical Fractionation; D018281:Cholangiocarcinoma / Q000737:chemistry / Q000175:diagnosis; D006801:Humans; D008113:Liver Neoplasms / Q000737:chemistry / Q000175:diagnosis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods
chemical_list: D014408:Biomarkers, Tumor; D020543:Proteome
keywords: Bile fluid; biomarkers; cholangiocellular carcinoma; hepatocellular carcinoma; method comparison; proteomics