Proteomic Profiling of Ovarian Cancer Plasma using Immunoaffinity Depleted Plasma and Two-Dimensional PAGE

Abstract

Abstract Objective The aim of this study was to evaluate a multiple immunoaffinity protein depletion (multiple affinity removal system, MARS) pre-treatment strategy with subsequent two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and peptide mass finger printing analysis for the detection of ovarian cancer-associated plasma proteins. Materials and Methods Following immunoaffinity depletion, total plasma protein content was reduced by 84.2 ± 1.8% (mean ± SE, n = 32). The number of proteins detected in the control and ovarian cancer groups was 349 and 357, respectively. This represented an increase in spot detection of almost twofold when compared to 2D PAGE displays of untreated plasma (174 spots). Of the proteins displayed, post-depletion, 300 (control) and 302 (ovarian cancer, OC) were common within each group. PDQuest analysis indicated that 109 protein spots were statistically different between the two groups and, of these, 59 exhibited greater than or equal to twofold difference in spot density (Student’s t test, p = 0.01). Thirty-nine of these proteins were successfully identified with reliable confidence. Results and Discussion The data obtained in this study demonstrates that immunodepletion of plasma before 2D PAGE profiling have generated identifiable plasma proteins that are differentially expressed in the high-grade ovarian cancer sample set compared to controls. This approach, therefore, may be useful in identifying candidate biomarkers for inclusion in multi-marker tests for ovarian cancer that may exhibit greater sensitivity and specificity than those currently available. It was evident, however, from the predominant identification of host response proteins that immunodepletion did not generate sufficient levels of enrichment of lower abundance tumor-specific proteins to facilitate detection.