Because serum is an optimal source to identify proteins derived from diseased-tissue compartments, serum proteomics has been applied for the discovery and analysis of biomarkers related to human disease. The general 2D-PAGE method is suitable for acidic and neutral proteome separation while highly basic proteins remain unresolved. In this study, we optimized basic proteome fractionation by integrating several methods such as, Micro-Rotofor isoelectric focusing (IEF) fractionation, acetic acid-urea polyacrylamide gel electrophoresis (A-U PAGE) separation, and mass spectrometry analysis for identifying basic proteins in patient sera. The sera samples were obtained from patients with atopic dermatitis (AD) to establish the model method introduced in the present study. We successfully identified the specific presence of a C3 complement component fragment protein and C4A fragment protein in AD patient-derived sera. Highly basic proteins in serum are very difficult to detect due to their separation properties. Thus, the integrated proteomic method approaches described here could be applicable for the detection of basic proteins associated with other diseases. (C) 2016 Elsevier Ltd. All rights reserved.