ARTICLE ABSTRACTThe use of advanced imaging technologies for the identification of pancreatic cysts has become widespread. However, accurate differential diagnosis between mucinous cysts (MC) and nonmucinous cysts (NMC) consisting of pseudocysts (NMC1) and nonmucinous neoplastic cysts (NMC2) remains a challenge. Thus, it is necessary to develop novel biomarkers for the differential diagnosis of pancreatic cysts. An integrated proteomics approach yielded differentially expressed proteins in MC that were verified subsequently in 99 pancreatic cysts (21 NMC1, 41 NMC2, and 37 MC) using a method termed GeLC-stable isotope dilution-multiple reaction monitoring-mass spectrometry (GeLC-SID-MRM-MS) along with established immunoassay techniques. We identified 223 proteins by nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC/MS-MS). Nine candidate biomarkers were identified, including polymeric immunoglobulin receptor (PIGR), lipocalin 2 (LCN2), Fc fragment of IgG-binding protein (FCGBP), lithostathine-1-alpha (REG1A), afamin (AFM), chymotrypsin C (caldecrin; CTRC), amylase, alpha 2B (pancreatic; AMY2B), lectin, galactoside-binding, soluble, 3 binding protein (LGALS3BP), and chymotrypsin-like elastase family, member 3A (CELA3A), which were established as biomarker candidates for MC. In particular, we have shown that a biomarker subset, including AFM, REG1A, PIGR, and LCN2, could differentiate MC not only from NMC (including NMC1) but also from NMC2. Overall, the MS-based comprehensive proteomics approach used in this study established a novel set of candidate biomarkers that address a gap in efforts to distinguish early pancreatic lesions at a time when more successful therapeutic interventions may be possible. Cancer Res; 75(16); 3227–35. ©2015 AACR.