This chapter discusses examples of methodology and considerations for examining the changes in protein abundances that cause and accompany acquired drug resistance in human breast cancer cells. The strategy includes isolation and examination of subcellular organelles, which provide smaller mixtures of proteins, and thus more total identifications; consideration of function within the context of organelle biology; and in many cases, new observations about which proteins are present or translocated in which cellular addresses. In the present work quantitative comparisons are made of the relative abundances of proteins in susceptible MCF-7 cells and a subline selected for resistance to mitoxantrone. Metabolic labeling with isotope-labeled arginine and lysine, and isotope ratio measurements made by matrix-assisted laser desorption ionization (MALDI) or electrospray mass spectrometry, were especially useful for difficult studies of plasma membrane proteins. A novel pellicle method was used to isolate enriched membrane proteins. Relative abundances of nuclear proteins were measured using metabolic labeling, and also by comparative densitometric analysis of twodimensional gel electrophoretic arrays. Around 5% of the proteins analyzed in each organelle were found to have abundances altered by more than twofold. Many of these were associated with the normal function of the organelle. Some correlations could be made between protein changes in the nucleus and membrane, notably in inhibition of apoptosis.
- Experimental design
- Organelle isolation
- Studies of drug resistance using organelle proteomics
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)