Abstract
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.
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