Use of narrow-range peptide IEF to improve detection of lung adenocarcinoma markers in plasma and pleural effusion

In this study we applied narrow-range peptide IEF to plasma or pleural effusion prior to LC/MS/MS. Two methods for narrow-range IEF were run; IPG strips and free-flow electrophoresis. Data from this study was compared with cell line data to evaluate the method performance in body fluids. To test the methods potential in quantitative biomarker discovery studies, plasma and pleural effusion from patients with lung adenocarcinoma (n = 3) were compared with inflammatory pleuritis (n = 3) using iTRAQ quantification. Using narrow-range IEF on the peptide level we were able to identify and quantify 282 proteins in plasma and 300 proteins in pleural effusion. These body fluid proteomes demonstrated high degree of overlap; however, more proteins significantly differently altered levels related to adenenocarcinoma were found in pleural effusion compared with plasma, suggesting enrichment of lung tissue-related proteins in pleural effusion. Nine proteins were chosen for initial validation with Western blot, and one protein (NPC2) was chosen for further validation using imunohistochemistry. Overall, the quantitative results from IEF/LC/MS/MS showed good correlation with the results from Western blot and imunohistochemistry, showing the potential of this methodology in quantitative biomarker discovery studies.