Proteome Analysis of a Breast Carcinoma Cell Line Overexpressing the HER2/neu Oncogene
EMSL Project ID
2608
Abstract
The application of stable-isotope labeling enables accurate quantitation of relative protein levels. A key aspect of our approach will be the use of isotopically labeled polypeptides, allowing two proteomes to be analyzed in the same separation. We will use the breast adenocarcinoma cell line MCF7 that has been engineered to overexpress the oncogene HER2/neu (MCF7 clone18). A two-pronged approach will be undertaken: isotope coded affinity tags (ICAT) and metabolic labeling with 15N enriched medium. ICAT entails separately labeling free thiol groups of cysteine residues (Cys) in proteins from two distinct populations of cells, with a biotinylated functionality for affinity purification and that has versions that differ in mass due to their isotopic content. For metabolic labeling of cellular proteins, MCF7clone18 will be grown in normal medium and MCF7neo will be grown in 15N-enrcihed medium. To measure changes in the relative expression of proteins, cells are harvested and mixed prior to sample processing and analysis. Combining the cells at this early time point will eliminate the experimental variables associated with cell lysis, separation, and MS analysis. This enables precise comparison of relative abundances for each protein since both labeled protein versions are extracted, digested and separated in an identical fashion. The proteins extracted from these combined populations will be digested with trypsin and analyzed at the peptide level by capillary LC-MS or LC-MS/MS. This approach, in combination with the high mass resolution, sensitivity, and dynamic range of FTICR, is expected to allow us to study changes in the expression of proteins that are undetectable by 2-D PAGE.
Project Details
Project type
Exploratory Research
Start Date
2002-09-25
End Date
2003-05-19
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members