Identification of heme binding protein complexes in murine erythroleukemic cells: study by a novel two-dimensional native separation -- liquid chromatography and electrophoresis |
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Authors: | Babusiak Marek Man Petr Sutak Robert Petrak Jiri Vyoral Daniel |
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Institution: | Institute of Hematology and Blood Transfusion, Prague, Czech Republic. marek.babusiak@uhkt.cz |
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Abstract: | 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 (59)Fe]-heme and cellular lysates were separated by anion-exchange chromatography. Fractions containing the (59)Fe 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. |
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Keywords: | [59Fe]hemin Heme binding protein Liquid chromatography Native electrophoresis Tandem mass spectrometry |
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