Quantitative chemical proteomics in small-scale culture of phorbol ester stimulated basal breast cancer cells.

Sibasish Dolai, Qiang Xu, Fei Liu, Mark P Molloy

Department of Chemistry and Biomolecular Sciences, Faculty of Science, Macquarie University, Sydney, Australia.

Proteomics 2011 Jul

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Basal-like breast cancers are commonly negative for expression of estrogen and progesterone receptors and HER-2 (triple-negative breast cancer), which makes this subtype of breast cancers more aggressive and less responsive to standard treatment. We have applied a small-scale chemical proteomics method using bisindolylmaleimide (Bis) class of protein kinase C inhibitors to study the Bis-binding proteome in a cell culture model of basal breast carcinoma (MDA-MB-231). Using MS, we identified 174 proteins captured by the Bis-probe in phorbol ester (PMA) stimulated cells. Gene ontology analysis broadly categorised these proteins as ATP binding (42%), GTP binding (6%) and having nucleoside-triphosphatase activity (21%). Of the 64 enzymes captured by the Bis-probe, the majority had either ATP and/or nucleotide binding functions. Two previously unreported Bis binding protein kinases, serine/arginine-rich protein-specific kinase 1 (SRPK1) and interferon-induced RNA-dependent protein kinase (PKR) were observed. We then incorporated SILAC for quantitation to examine the proteins that were differentially captured by the Bis-probe following 30 and 60 min PMA stimulation. This provided novel evidence for PMA regulation of the enzymes glyceraldehyde-3-phosphate dehydrogenase, nucleolar RNA helicase 2 and Heterogeneous nuclear ribonucleoprotein M. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Citation

Sibasish Dolai, Qiang Xu, Fei Liu, Mark P Molloy. Quantitative chemical proteomics in small-scale culture of phorbol ester stimulated basal breast cancer cells. Proteomics. 2011 Jul;11(13):2683-92