Quantitative Organelle Proteomics of MCF-7 Breast Cancer Cells Reveals Multiple Subcellular Locations for Proteins in Cellular Functional Processes

Abstract

We have combined sucrose density gradient subcellular fractionation with quantitative, tandemmass-spectrometry-based shotgun proteomics to investigate spatial distributions of proteins in MCF-7 breast cancer cells. Emphasis was placed on four major organellar compartments: cytosol, plasma membrane, endoplasmic reticulum, and mitochondrion. Two-thousand one-hundred eighty-four proteins were securely identified. Four-hundred eighty-one proteins (22.0% of total proteins identified) were found in unique sucrose gradient fractions, suggesting they may have unique subcellular locations. 454 proteins (20.8%) were found to be ubiquitously distributed. The remaining 1249 proteins (57.2%) were consistent with intermediate distribution over multiple, but not all, subcellular locations. Ninety-four proteins implicated in breast cancer and 478 other proteins which share the same five major cellular biological processes with a majority of the breast cancer proteins were observed in 334 and 1223 subcellular locations, respectively. The data obtained is used to evaluate the possibility of defining more exact sets of subcellular organelles, the completeness of current descriptions of spatial distribution of cellular proteins, the importance of multiple subcellular locations for proteins in functional processes, the subcellular distribution of proteins related to breast cancer, and the possibility of using these methods for dynamic spatio/ temporal studies of function/regulation in MCF-7 breast cancer cells.

Spatial Distribution of Cellular Function: The Partitioning of Proteins between Mitochondria and the Nucleus in MCF7 Breast Cancer Cells

“…The pellet was air-dried for 5 min to eliminate any acetone residue and then resuspended in a 1× protein solubilization buffer (10 mM PIPES pH 7.3, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid). 38 2.4. Preparation of Mitochondrial Proteins.…”

“…Samples were reduced by 10 mM dithiothreitol DTT and alkylated with 100 mM iodoacetamide by use of the ProGest Investigator Instrument (DigiLab, Genomics Solutions, Cambs, U.K.) according to the established protocol. 38 Subsequently, 200 ng of sequencing grade modified trypsin (Promega, Hamps, U.K.) in 50 mM ammonium bicarbonate was added to dry gel pieces and the mixture incubated overnight at 37 °C as described previously. 46 Tryptic peptides were eluted from the gel, concentrated and dissolved in 0.1% formic acid for LC-MS/MS analysis.…”

“…According to our established protocol as described previously, 38 peptide samples were analyzed with a LTQ-Orbitrap-XL MS (Thermo Fisher Scientific, Surrey, U.K.), using a capillary column, NTCC-360/100-5-153 (100 μm I.D, 5 μm C18 particles, 15 cm length, Nikkyo Technos CO, Tokyo, Japan) and a nanoelectrospray ion source at a flow rate reduced via a splitter of 0.9 μL/min. The mobile phase comprised 0.1% formic acid (Solvent A) and 100% acetonitrile with 0.1% formic acid (solvent B).…”

“…38 M, S, R and G are all daughters of cytoplasm in the GO ontology and the cytoplasm annotation was therefore used only when no low level annotation was available. Overall, 36% of the proteins in the full GO CC human set had annotations to more than one subcellular location with this classification scheme.…”

“…We recently used proteomics methods to show that at least 50% and perhaps as much as much as 75% of proteins detected in MCF7 cells show multiple locations involving significant proportions of each protein. 38 In yeast, recent studies indicate that at least 30% of mitochondrial proteins also have other subcellular locations. 39 In the context of nuclear import/export, there has long been evidence that dynamic relocation of proteins to and from the nucleus is crucial to cellular function and there is increasing evidence for changes in nuclear transport following perturbations such as oxidative stress.…”