Subcellular fractionation enhances proteome coverage of pancreatic duct cells.

OBJECTIVES: Subcellular fractionation of whole cell lysates offers a means of simplifying protein mixtures, potentially permitting greater depth of proteomic analysis. Here we compare proteins identified from pancreatic duct cells (PaDC) following organelle enrichment to those identified from PaDC whole cell lysates to determine if the additional procedures of subcellular fractionation increase proteome coverage. METHODS: We used differential centrifugation to enrich for nuclear, mitochondrial, membrane, and cytosolic proteins. We then compared - via mass spectrometry-based analysis - the number of proteins identified from these four fractions with four biological replicates of PaDC whole cell lysates. RESULTS: We identified similar numbers of proteins among all samples investigated. In total, 1658 non-redundant proteins were identified in the replicate samples, while 2196 were identified in the subcellular fractionation samples, corresponding to a 30% increase. Additionally, we noted that each organelle fraction was in fact enriched with proteins specific to the targeted organelle. CONCLUSIONS: Subcellular fractionation of PaDC resulted in greater proteome coverage compared to PaDC whole cell lysate analysis. Although more labor intensive and time consuming, subcellular fractionation provides greater proteome coverage, and enriches for compartmentalized sub-populations of proteins. Application of this subcellular fractionation strategy allows for a greater depth of proteomic analysis and thus a better understanding of the cellular mechanisms of pancreatic disease.

Analysis of endoscopic pancreatic function test (ePFT)-collected pancreatic fluid proteins precipitated via ultracentrifugation.

CONTEXT: We have shown previously that trichloroacetic acid precipitation is an effective method of protein extraction from pancreatic fluid for downstream biomarker discovery, compared to other common extraction methods tested. OBJECTIVE: We aim to assess the utility of ultracentrifugation as an alternative method of protein extraction from pancreatic fluid. DESIGN: Proteins extracted from trichloroacetic acid- and ultracentrifugation-precipitated pancreatic fluid were identified using mass spectrometry techniques (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry; GeLC-MS/MS). Data were analyzed using Proteome Discoverer and Scaffold 3. SETTING: This is a proteomic analysis experiment of endoscopically collected fluid in an academic center. PATIENTS: The study population included adult patients referred to the Center for Pancreatic Disease at Brigham and Women's Hospital, Boston, MA, USA for the evaluation of abdominal pain and gastrointestinal symptoms. INTERVENTIONS: Secretin-stimulated pancreatic fluid was collected as standard of care for the evaluation of abdominal pain and gastrointestinal symptoms. MAIN OUTCOME MEASURES: We compared proteins identified via standard trichloroacetic acid precipitation and this alternative ultracentrifugation strategy. RESULTS: A subset of pancreatic fluid proteins was identified via the ultracentrifugation method. Of these proteins, similar numbers were obtained from fully tryptic or semi-tryptic database searching. Proteins identified in the ultracentrifugation-precipitated samples included previously identified biomarker candidates of chronic pancreatitis. CONCLUSIONS: This alternative ultracentrifugation strategy requires less time and fewer handling procedures than standard trichloroacetic acid precipitation, at the expense of higher sample volume. As such, this method is well suited for targeted assays (i.e., dot blotting or targeted mass spectrometry) if the protein of interest is among those readily identified by ultracentrifugation-promoted precipitation.