MCF-7 Cell Fractionation and Proteomics

MCF-7 cells (ATCC, Manassas, VA) were grown in EMEM with 10% FBS and 10 μg/mL bovine insulin, in an incubator at 37°C with 5% CO₂[7 (link), 8 (link)]. The cells were arrested in the G1-phase by serum-deprivation for 48 h, in a medium consisting of DMEM and 4 mM L-glutamine, harvested, separated into nuclear and cytoplasmic fractions (Cell Lytic™ NuCLEAR™ extraction kit, Sigma, St. Louis, MO), digested with trypsin (Promega Corporation (Madison, WI) at 37°C for 24 h (50:1 substrate:enzyme ratio), and analyzed by nano-liquid chromatography (LC)-MS/MS with a linear trap quadrupole (LTQ/Thermo Electron Corporation, San Jose, CA) mass spectrometer. FACS analysis was performed with a Beckman Coulter EPICS XL-MCL analyzer (Brea, CA, USA). The protein content was measured by the Bradford assay on a SmartSpec Plus spectrophotometer (Bio-Rad, Hercules, CA). The sample analyzed by MS contained 2 μg/μL MCF-7 proteins. LC separations were performed with an Agilent 1100 LC system (Palo Alto, CA) and in-house prepared nano-separation columns (100 μm i.d. x 12 cm) packed with 5 μm Zorbax SB-C18 particles. Common reagents were purchased from Sigma, cell culture media from ATCC and Invitrogen (Carlsbad, CA), and HPLC-solvents from Fisher Scientific (Fair Lawn, NJ). Sample preparation and LC-MS/MS analysis protocols were described in detail in previous manuscripts [7 (link), 8 (link)].