Breast cancer protein StarD10 identified by three-dimensional separation using free-flow electrophoresis, reversed-phase high-performance liquid chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
A 35 kDa protein present in mammary tumors from Neu/ErbB2 transgenic mice was detected on the basis of its cross-reactivity with a phosphoserine-specific antibody against the transcription factor FKHR. To isolate this protein from cytosolic extracts derived from human breast carcinoma cells, we used free-flow electrophoresis in the first dimension to separate proteins according to their charge, followed by reversed-phase high-performance liquid chromatography (RP-HPLC) in the second and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the third dimension. Tryptic digests of Coomassie-stained bands were analyzed by nano-spray ionization-quadrupole quadrupole-time of flight-mass spectrometry identifying StarD10, a START domain containing protein, which cross-reacted with the anti-phospho-FKHR antibody. The site of phosphorylation was identified in immunoaffinity purified Flag-tagged StarD10 from 293T cells transiently expressing this protein. Tryptic phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC) and StarD10 Ser-259-phosphate was identified by tandem mass spectrometry. Thus, free-flow electrophoresis is a powerful high-capacity complementary technique to RP-HPLC and SDS-PAGE for the purification of proteins from complex cell lysates.
Hoffmann
Peter
P
St. Vincent's Institute and CSIRO Health Sciences & Nutrition, Victoria, Australia. peter.hoffmann@bbz.uni-leipzig.de
Olayioye
Monilola A
MA
Moritz
Robert L
RL
Lindeman
Geoffrey J
GJ
Visvader
Jane E
JE
Simpson
Richard J
RJ
Kemp
Bruce E
BE
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003429:Cross Reactions; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008325:Mammary Neoplasms, Experimental / Q000737:chemistry; D051379:Mice; D008822:Mice, Transgenic; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000302:isolation & purification*; D010766:Phosphorylation; D021241:Spectrometry, Mass, Electrospray Ionization; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism
D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D010750:Phosphoproteins; C488091:Stard10 protein, mouse; D014157:Transcription Factors