A proteomic comparison of formalin-fixed paraffin-embedded pancreatic tissue from autoimmune pancreatitis, chronic pancreatitis, and pancreatic cancer.

CONTEXT: Formalin-fixed paraffin-embedded (FFPE) tissue is a standard for specimen preservation, and as such FFPE tissue banks are an untapped resource of histologically-characterized specimens for retrospective biomarker investigation for pancreatic disease. OBJECTIVES: We use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to compare FFPE specimens from three different diseases of the exocrine pancreas. DESIGN: We investigated the proteomic profile of FFPE pancreatic tissue from 9 archived specimens that were histologically classified as: autoimmune pancreatitis (n=3), chronic pancreatitis (n=3), and pancreatic cancer (n=3), using LC-MS/MS. SETTING: This is a proteomic analysis experiment of FFPE pancreatic tissue in an academic center. PATIENTS: FFPE tissue specimens were provided by Dana-Farber/Harvard Cancer Center (Boston, MA, USA). INTERVENTIONS: FFPE tissue specimens were collected via routine surgical resection procedures. MAIN OUTCOME MEASURES: We compared proteins identified from chronic pancreatitis, autoimmune pancreatitis, and pancreatic cancer FFPE pancreatic tissue. RESULTS: We identified 386 non-redundant proteins from 9 specimens. Following our filtering criteria, 73, 29, and 53 proteins were identified exclusively in autoimmune pancreatitis, chronic pancreatitis, and pancreatic cancer specimens, respectively. CONCLUSIONS: We report that differentially-expressed proteins can be identified among FFPE tissues specimens originating from individuals with different histological diagnoses. These proteins merit further confirmation with a greater number of specimens and orthogonal validation, such as immunohistochemistry. The mass spectrometry-based methodology used herein has the potential to enhance diagnostic biomarker and therapeutic target discovery, further advancing pancreatic research.

Post-translational modifications of pancreatic fluid proteins collected via the endoscopic pancreatic function test (ePFT).

BACKGROUND: Early diagnosis of chronic pancreatitis by mass spectrometry-based proteomics may result in therapies to retard or modify disease progression. We aimed to identify differences in posttranslational modifications (PTMs) in pancreatic fluid proteins from individuals with chronic pancreatitis (n=9) and non-pancreatitis controls (n=9). METHODS: We collected proteomic data from pancreatic fluid using mass spectrometry techniques. We performed database searches with emphasis on PTMs using ProteinPilot. We compared the frequency of specific PTMs in pancreatic fluid between cohorts and also to those identified in bile, gastroduodenal fluid, urine, and pancreatic duct and stellate cell lysates. RESULTS: We identified 97 PTMs in endoscopically-collected pancreatic fluid, of which 11 were identified exclusively in one cohort and 9 others were significantly different in frequency between cohorts. Comparing pancreatic fluid with other specimens revealed differences in specific PTM frequencies, indicating that the identified PTMs were not merely artifacts of sample processing. CONCLUSIONS: We determined PTMs of proteins extracted from pancreatic fluid which differed in frequency in chronic pancreatitis patients verses controls. Such PTMs may serve as biomarker candidates of chronic pancreatitis upon validation with larger cohorts. The analysis of the PTM profile of pancreatic fluid proteins offers an alternative method to standard protein-based biomarker discovery. BIOLOGICAL SIGNIFICANCE: The early diagnosis of chronic pancreatitis is paramount in developing strategies to modify, retard, or halt disease progression. In the present study, we compared post-transitional modifications (PTMs) of proteins extracted from pancreatic fluid of chronic pancreatitis patients verses a control cohort. With many mass spectrometry-based proteomics workflows aimed to identify and quantify proteins, data for PTMs typically comes gratis, in that such data are collected during protein sequencing and, as such, require only downstream bioinformatics processing. We identified a total of 20 PTMs which were exclusive to or significantly different between cohorts. Upon validation with larger cohorts and enrichment of these PTMs may serve as biomarker candidates of chronic pancreatitis. PTM profiling of pancreatic fluid proteins is complementary to standard protein-based biomarker discovery, and may be readily applied to studies of pancreatic disease. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Short Gel, Long Gradient Liquid Chromatography Tandem Mass Spectrometry to Discover Urinary Biomarkers of Chronic Pancreatitis.

BACKGROUND: Chronic pancreatitis (CP) is currently diagnosed using invasive endoscopic as well as radiation and non-radiation-based imaging techniques. However, urine can be safely and non-invasively collected and as such may offer a superior alternative to current techniques of CP diagnosis. We use mass spectrometry-based methods to discover proteins which are exclusive to or differentially abundant in urine of chronic pancreatitis patients. METHODS: We have performed a comparative quantitative proteomic analysis of urine collected from 5 healthy controls and 5 severe CP patients. Proteins from urine were fractionated briefly on SDS-PAGE and subsequently digested in-gel with trypsin. The resulting peptides were fractionated for 3 hours by reversed-phase liquid chromatography in-line with a mass spectrometer. ProteinPilot software and the QSPEC algorithm identified proteins and determined statistically significant differences between cohorts. In addition, we used a third cohort of non-CP disease patients to filter out those proteins which may be indicative of an ailment other than CP. RESULTS: We identified over 600 proteins from urine, of which several hundred were either exclusive to or differ quantitatively between healthy controls and severe CP patients. Members of the cathepsin protein family were of significantly higher abundance in the severe CP cohort. In addition, we have identified a core set of 50 proteins in all 15 samples, 25 of which showed no significant difference among the cohorts. CONCLUSIONS: Proteomic analysis identified differentially abundant proteins in healthy controls and severe CP patients. Such proteins represent an initial set of targets for directed proteomics experiments for further validation studies. However, larger cohorts will be required to determine if these differences have statistically significant diagnostic potential.