Identification of heme binding protein complexes in murine erythroleukemic cells: study by a novel two-dimensional native separation -- liquid chromatography and electrophoresis.

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.

• Authors: Marek Babusiak, Petr Man, Robert Sutak, Jiri Petrak, Daniel Vyoral
• Journal: Proteomics (Proteomics) Vol. 5 Issue 2 Pg. 340-50 (Feb 2005) ISSN: 1615-9853 [Print] Germany
• PMID: 15627969 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Carrier Proteins
• Heme-Binding Proteins
• Hemeproteins
• Hemoglobins
• Iron Radioisotopes

Topics

• Animals
• Carrier Proteins (analysis)
• Cell Line, Tumor
• Chromatography, Ion Exchange
• Chromatography, Liquid
• Electrophoresis, Gel, Two-Dimensional
• Electrophoresis, Polyacrylamide Gel
• Heme-Binding Proteins
• Hemeproteins (analysis)
• Hemoglobins (biosynthesis)
• Iron Radioisotopes (metabolism)
• Leukemia, Erythroblastic, Acute (metabolism)
• Mass Spectrometry
• Mice
• Models, Biological