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 [(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.
Babusiak
Marek
M
Institute of Hematology and Blood Transfusion, Prague, Czech Republic. marek.babusiak@uhkt.cz
Man
Petr
P
Sutak
Robert
R
Petrak
Jiri
J
Vyoral
Daniel
D
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D045744:Cell Line, Tumor; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000032:analysis*; D006454:Hemoglobins / Q000096:biosynthesis; D007504:Iron Radioisotopes / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D008954:Models, Biological
D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D006454:Hemoglobins; D007504:Iron Radioisotopes