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 Flagtagged StarD10 from 293T cells transiently expressing this protein, Tryptic phosphopepticles were enriched by immobilized metal affinity chromatography (IMAC) and StarD10 Ser-259-phosphate was identified by tandem mass spectrometry. Thus, freeflow electrophoresis is a powerful high-capacity complementary technique to RPHPLC and SIDS-PAGE for the purification of proteins from complex cell lysates.