IDENTIFICATION OF UNIQUE DIFFERENTIAL PROTEOMIC BIOMARKERS IN PANCREATIC CYSTIC NEOPLASM FLUID

Abstract

Background: Current preoperative differentiation of premalignant and malignant pancreatic cysts from benign pancreatic cysts based on imaging and cyst fluid analysis is inadequate. This inability to accurately differentiate has resulted in unnecessary surgical resections. This is the first study applying proteomic technology to identify novel proteins or protein patterns in pancreatic cyst fluid which may more accurately predict pancreatic cyst pathology preoperatively and help triage patient care. Methods: Pancreatic cyst fluid was collected by endoscopic ultrasound guided FNA from patients with pathologically confirmed pancreatic mucinous and non-mucinous cysts, centrifuged and stored immediately at −80°C. Fluid from representative pancreatic cysts was prepared for proteomic analysis using centrifugation, acetone, ethanol and TCA precipitation, followed by solubilization into highly chaotropic nonionic detergents and buffer for 2-D electrophoresis and 2 dimensional liquid chromatography (PF2D: 1-D chromatofocussing, 2D-reverse phase). Spots from 2D gels were robotically excised, digested and mass spectrometry was used to identify unique differential proteins using liquid chromatography tandem mass spectrometry (LC-MS/MS) and MALTI-TOF. Proteins were identified using Mascot search engine and Sequest search algorithms. Results: 122 pancreatic cyst fluid samples were collected, stored and processed according to the protocol. Initial protein quantification for ranged from 1 to 5 mg/ml. Pancreatic cyst fluid from 5 samples (2 mucinous and 3 serous cystadenomas) was used for initial proteomic analysis (2D gel electrophoresis (2DE:(3)), PF2D(2)). Initial analysis shows a striking differential protein profile between serous and mucinous samples (by PF2D) in both dimensions (1-D chromatofocussing and 2-D reverse phase). Comparison of two-dimensional maps of the pancreatic serous and mucinous cystadenomas show few overlaps thus suggesting many proteins unique to each cystic neoplasm. Interpretable 2-D electrophoresis gels were obtained from pancreatic cyst fluid. Using 2-D Electrophoresis we have already identified over 20 unique cellular and membrane proteins using LC-MS/MS. Through 2-D electrophoresis gel we have identified cellular and membrane proteins using LC-MS/MS. Chromatofocussed fractions were processed over a second dimension HPRP column to produce over 700 fractions in each experiment. The second dimension reverse phase fractions for serous cyst show a very different protein profile than mucinous cysts. Conclusions: To our knowledge, this is the first report detailing the proteome of pancreatic cystic neoplasm fluid. Differential protein expression was found between pancreatic mucinous and serous cystadenoma fluid. Identification of specific protein patterns or proteins may be used as highly accurate serum based or cyst fluid based biomarkers.