A Mammalian Organelle Map by Protein Correlation Profiling

Foster, Leonard J.; Hoog, Carmen L. de; Zhang, Yanling; Zhang, Yong; Xie, Xiaohui; Mootha, Vamsi K.; Mann, Matthias

doi:10.1016/j.cell.2006.03.022

Cited by 547 publications

(586 citation statements)

References 43 publications

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“…On the basis of work previously published by us 38 and others, 51,52 we used subfractionation of cellular organelles by sucrose gradient centrifugation. The protein samples from nuclear (N) and mitochondrial (M) fractions of sucrose gradient subcellular fractionations of MCF7 cells were further fractionated by SDS gel electrophoresis or pI prior to trypsin treatment and MS analyses.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2012

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Section: Resultsmentioning

confidence: 99%

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

et al. 2012

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“…Our use of subfractionation of cellular organelles by sucrose gradient centrifugation is based on previous proteomics work indicating that fractionation of cytosol, plasma membrane, endoplasmic reticulum, Golgi and mitochondria is readily obtained 22 and that with more sophisticated analysis of protein distribution along the gradient, as many as 10 subcellular locations can be distinguished. 23 The subsequent steps in identifying the proteins present in different fractions of the sucrose gradient (1D SDS gels, gel slicing, proteolytic production of peptides, and identification of peptides by MS) are standard proteomics techniques, and our use of them is described in detail in the Materials and Methods section. We note that the MUDPIT approach 15 has been used in connection with a fused silica C18 capillary column for elution and a nanoelectrospray ion source.…”

Section: Resultsmentioning

confidence: 99%

“…The main disadvantages of this approach are that the degree of purification/ contamination of the organelle is difficult to ascertain conclusively for lower abundance proteins, that the protein content may be altered by the purification process and that the approach is not very suitable for dynamic studies of protein subcellular location. In a few cases, 22,23 an alternative approach of partial purification of organelles in a sucrose gradient has been employed, but the assignment of proteins to individual organelles has been based on matching gradient profiles of proteins to the profiles of presumptive marker proteins. This is useful for identifying what might be denominated core proteins of an organelle, but is automatically biased against evaluation of proteins in multiple subcellular locations.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2009

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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.

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“…Several groups have utilized label-free quantitative proteomics in the high throughput assignment of proteins to subcellular compartments. In one approach, protein correlation profiling, proteins from enriched organelle fractions are quantified by peptide ion intensity measurements (3,4). Other similar methods employ quantitation by spectral counting, recording the number of ions detected per protein (5,6).…”

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The Organelle Proteome of the DT40 Lymphocyte Cell Line

Hall

Hester

Griffin

et al. 2009

Molecular & Cellular Proteomics

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A major challenge in eukaryotic cell biology is to understand the roles of individual proteins and the subcellular compartments in which they reside. Here, we use the localization of organelle proteins by isotope tagging technique to complete the first proteomic analysis of the major organelles of the DT40 lymphocyte cell line. This cell line is emerging as an important research tool because of the ease with which gene knockouts can be generated. We identify 1090 proteins through the analysis of preparations enriched for integral membrane or soluble and peripherally associated proteins and localize 223 proteins to the endoplasmic reticulum, Golgi, lysosome, mitochondrion, or plasma membrane by matching their density gradient distributions to those of known organelle residents. A striking finding is that within the secretory and The chicken pre-B cell line DT40 exhibits a remarkably high ratio of targeted to random integration for transfected DNA constructs. This property is unusual in vertebrate cell lines and enables targeted gene disruption experiments to be carried out with relative ease (1). Consequently, DT40 has become a major research tool for the molecular dissection of a wide range of cellular and biochemical mechanisms in a vertebrate context, including membrane traffic, signal transduction, and cell cycle (2).Proteins in eukaryotic cells are organized according to their functions within a dynamic network of membranes. Localization is therefore paramount in assigning functions to uncharacterized proteins and understanding the processes occurring in subcellular compartments. An increased knowledge of the protein localization within the DT40 cell line would be of great value. Traditional localization methods such as immunofluorescence microscopy are typically low throughput and are more suitably applied to the study of specific proteins of interest rather than the cataloguing of large numbers of proteins. Recent developments in proteomics have made it possible to analyze the protein composition of organelles using a variety of different approaches. Several groups have utilized label-free quantitative proteomics in the high throughput assignment of proteins to subcellular compartments. In one approach, protein correlation profiling, proteins from enriched organelle fractions are quantified by peptide ion intensity measurements (3, 4). Other similar methods employ quantitation by spectral counting, recording the number of ions detected per protein (5, 6). Localization of organelle proteins by isotope tagging (LOPIT) 1 is a complementary approach, which employs isotope labeling for quantitation (7-9). Rather than processing each sample separately as in label-free techniques, differentially labeled fractions are pooled early in the LOPIT protocol. This has the important advantage of reducing the points at which variation might be introduced into the data.LOPIT begins with the partial separation of organelles by density gradient centrifugation and relies on the assumption that proteins from each organelle c...

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A Mammalian Organelle Map by Protein Correlation Profiling

Cited by 547 publications

References 43 publications

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

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

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

The Organelle Proteome of the DT40 Lymphocyte Cell Line

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