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Blood, 15 May 2004, Vol. 103, No. 10, pp. 3751-3759.
Prepublished online as a Blood First Edition Paper on February 5, 2004; DOI 10.1182/blood-2003-09-3294.
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HEMATOPOIESIS
Comparative proteomics of primitive hematopoietic cell populations reveals differences in expression of proteins regulating motility
Caroline A. Evans,
Robert Tonge,
David Blinco,
Andrew Pierce,
Joanne Shaw,
Yuning Lu,
Hajja G. Hamzah,
Alexander Gray,
C. Peter Downes,
Simon J. Gaskell,
Elaine Spooncer, and
Anthony D. Whetton
From the Leukaemia Research Fund Proteomics Facility, UMIST; LRF Cellular Development Unit, UMIST; Department of Biomolecular Sciences, UMIST; Michael Barber Centre for Mass Spectrometry, Department of Chemistry, University of Manchester Institute of Science and Technology (UMIST), Manchester, United Kingdom; Global Protein Science and Supply, Enabling Science and Technology (Biology), AstraZeneca, Cheshire, United Kingdom; and the School of Life Sciences, MSI/WTB Complex, University of Dundee, United Kingdom.
Lineage-marker depleted (Lin–) murine bone marrow cells expressing stem cell antigen 1 (Sca-1) were sorted on the basis of stem cell factor receptor (c-kit) expression to obtain Lin–Sca+Kit+ or Lin–Sca+Kit– cells. Lin–Sca+Kit– cells have a markedly greater chemotactic response to stromal derived factor-1 (SDF-1). Using a novel fluorescent stain, we show that both populations generate similar levels of a key messenger, phosphatidylinositol 3,4,5 trisphosphate (PIP3), in response to SDF-1. Differences in motile behavior may therefore lie downstream of phosphatidylinositol 3-kinase (PI3-kinase) activation at the level of cytoskeleton regulation. The 2 cell populations were compared using 2-dimensional difference gel electrophoresis (2D-DIGE), with a maleimide CyDye fluorescent protein labeling technique that has enhanced sensitivity for low abundance samples. Comparative proteomic analysis of Cy3- and Cy5-labeled protein samples allows relative quantification of protein spots present in both cell populations; of these, 73% were common. Key protein differences were adseverin and gelsolin, actin micro-filament splicing proteins, regulated by Rac, downstream of PI3-kinase activation. Adseverin was shown to be acetylated, a novel modification for this protein. Differences in major regulators of cell shape and motility between the 2 populations can explain the differential response to SDF-1.
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