Hydrophobicity and molecular mass-based separation method for autoantibody discovery from mammalian total cellular proteins

Serum autoantibody profiles are unique to individuals and reflect the level and history of autoimmunity and tumor immunity. The identification of autoantibody biomarkers is critical for the development of immune monitoring systems for immune-related disorders. Here, we present a practical method for large-scale autoantibody discovery using total cellular proteins from cultured mammalian cells. We found that nucleic acid-free and fully denatured water-soluble total cellular proteins from mammalian cells were superior, allowing precise separation by reversed-phase HPLC after preparing a large set of homogeneous total cellular proteins. After separating the proteins based on hydrophobicity, the fractionated samples were subjected to molecular mass analysis using conventional SDS-PAGE. The resulting two-dimensional gel electrophoresis was successfully employed for immune blotting and LC–MS/MS analysis. All procedures, including TRIzol-based total cellular protein extraction, solubilization of denatured proteins, reversed-phase HPLC separation, and SDS-PAGE, were highly reproducible and easily scalable. We propose this novel two-dimensional gel electrophoresis system as an alternative proteomics-based methodology suitable for large-scale autoantibody discovery.