Assessing the phosphorylation status of estrogen receptor alpha isolated from human breast cancer cells

摘要

Activated estrogen receptor alpha (ERa) is of critical importance in the initiation/promotion mechanisms of most human breast cancers, and serves as the molecular target for anti-hormonal therapies. Phosphorylation of ERa is known to induce significant ERa activation and also potential resistance mechanisms to anti-hormonal therapies. Biochemical methods of phosphorylation detection using specific antibodies have practical limitations, including a presupposed knowledge of the PTM site(s) of interest and the availability/quality of the requisite site-specific antibody. Therefore a combination of ESI-MS/MS (Q-STAR/4000 Q-TRAP) and vMALDI-MS~(n) (vMALDI-LTQ) was employed to identify novel and confirm known phosphorylation sites of commercially available human ERa expressed in insect cells (rERa), and endogenously expressed ERa immunoprecipitated from control and estradiol treated human breast cancer cells (MCF-7).