title: Quantitative chemical proteomics in small-scale culture of phorbol ester stimulated basal breast cancer cells.
abstract: 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.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Faculty of Science, Macquarie University, Sydney, Australia.
authors.forename: Sibasish
authors.initials: S
authors.lastname: Dolai
authors.forename: Qiang
authors.initials: Q
authors.lastname: Xu
authors.forename: Fei
authors.initials: F
authors.lastname: Liu
authors.forename: Mark P
authors.initials: MP
authors.lastname: Molloy
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D007211:Indoles / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D015394:Molecular Structure; D018295:Neoplasms, Basal Cell / Q000378:metabolism* / Q000473:pathology; D010703:Phorbol Esters / Q000494:pharmacology*; D040901:Proteomics / Q000379:methods*; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism
chemical_list: D007211:Indoles; D008301:Maleimides; D010703:Phorbol Esters; C088060:bisindolylmaleimide