Identification of heme binding protein complexes in murine erythroleukemic cells: Study by a novel two‐dimensional native separation – liquid chromatography and electrophoresis

In the current postgenomic era there is a growing interest in analysis of protein complexes in their native state. Here we present a novel two‐dimensional separation technique for assessment of native protein complexes. The method combines native chromatography with native electrophoresis. The approach was used to study heme‐binding protein complexes in murine erythroleukemia cells. The cells were metabolically labeled with [59Fe]‐heme and cellular lysates were separated by anion‐exchange chromatography. Fractions containing the 59Fe isotope were collected, concentrated and further separated by native gel electrophoresis. A total of 13 radioactive protein bands were detected and analyzed by liquid chromatography‐tandem mass spectrometry. Thirty‐three individual proteins were identified and attributed to four novel multiprotein complexes representing four different ‘snapshots’ of cellular events involved in hemoglobin biosynthesis.