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https://hdl.handle.net/2440/34805
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Type: | Journal article |
Title: | 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 |
Author: | Hoffmann, P. Olayioye, M. Moritz, R. Lindeman, G. Visvader, J. Simpson, R. Kemp, B. |
Citation: | Electrophoresis, 2005; 26(6):1029-1037 |
Publisher: | Wiley-V C H Verlag GMBH |
Issue Date: | 2005 |
ISSN: | 0173-0835 1522-2683 |
Statement of Responsibility: | Peter Hoffmann, Monilola A. Olayioye, Robert L. Moritz, Geoffrey J. Lindeman, Jane E. Visvader, Richard J. Simpson, Bruce E. Kemp |
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. |
Keywords: | Immobilized metal ion affinity chromatography Serine phosphorylation START domain Tandem mass spectrometry |
Description: | The definitive version may be found at www3.interscience.wiley.com |
DOI: | 10.1002/elps.200406197 |
Published version: | http://dx.doi.org/10.1002/elps.200406197 |
Appears in Collections: | Aurora harvest 6 Molecular and Biomedical Science publications |
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