Key Points
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Proteomics techniques have been successfully used to identify the protein components of organelles and large cellular structures.
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Several strategies have been developed that can be used to separate and identify the proteins in enriched organelle or subcellular fractions, and these include two-dimensional gel electrophoresis, one-dimensional SDS-PAGE and multidimensional liquid-chromatography–tandem mass spectrometry.
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Subtractive proteomics methods have been effective in highlighting the proteins that are specifically localized to organelles and other structures. However, it is difficult to identify the appropriate controls for these methods.
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Significant proteomics data have been accumulated for important organelles, such as mitochondria, the Golgi complex, the nuclear envelope, the endoplasmic reticulum and phagosomes.
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Large cellular structures such as the midbody, cytoskeleton and lipid rafts have also been successfully analysed using proteomics techniques.
Abstract
The mass-spectrometry-based identification of proteins has created opportunities for the study of organelles, transport intermediates and large subcellular structures. Traditional cell-biology techniques are used to enrich these structures for proteomics analyses, and such analyses provide insights into the biology and functions of these structures. Here, we review the state-of-the-art proteomics techniques for the analysis of subcellular structures and discuss the biological insights that have been derived from such studies.
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Acknowledgements
We would like to acknowledge support from the National Institutes of Health (J.R.Y. and K.E.H.), Genome Canada/Genome Québec (J.J.M.B.), Valorisation Recherche Québec (J.J.M.B.), and the Canada Foundation for Innovation: Cell Map Project (J.J.M.B.).
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MASS SPECTROMETRY COMPANIES
Glossary
- CAVEOLAE
-
A plasma-membrane invagination that is enriched in caveolin, cholesterol and glycosphingolipids.
- EXOSOMES
-
Small vesicles of endocytic origin that are secreted by various cell types
- LC–MS/MS
-
(liquid-chromatography–tandem mass spectrometry). This process uses an instrument in which high-performance liquid chromatography is linked directly to a tandem mass spectrometer through electrospray ionization.
- COLLISION-INDUCED DISSOCIATION
-
A process that is used in tandem mass spectrometry to cause ions to fragment and reveal information about their molecular structure. Peptides fragment at their amide bonds to produce a ladder of sequence ions that are representative of the amino-acid sequence of the peptide.
- MUDPIT
-
(multidimensional protein-identification technology). A method to identify the proteins that were digested to form peptide mixtures. The method uses multidimensional liquid chromatography together with tandem mass spectrometry and database searching.
- STRONG CATION-EXCHANGE RESIN
-
An ion-exchange resin with a negatively charged surface that binds positively charged ions.
- REVERSED-PHASE SUPPORT
-
A chromatographic resin that binds molecules on the basis of their hydrophobicity. It is often used for the separation of peptides.
- PHAGOSOME
-
A membrane-bounded intracellular vesicle that arises from the ingestion of particulate material by phagocytosis.
- PHAGOLYSOSOME
-
A phagocytic vesicle that has fused with a lysosome to become a digestive vacuole.
- QUADRUPOLE TIME-OF-FLIGHT–MS/MS
-
(quadrupole time-of-flight–tandem mass spectrometry). A hybrid tandem-mass-spectrometry technique that combines a quadrupole mass analyser (which uses oscillating electric fields on four metal rods to separate ions on the basis of their mass-to-charge ratio) with a time-of-flight mass analyser (which converts the time it takes an ion to travel a specific distance into a mass-to-charge ratio).
- DIGE
-
(fluorescence difference two-dimensional (2D) gel electrophoresis). A form of 2D gel electrophoresis that uses different colour fluorescent dyes to label proteins from different states that are then mixed and separated. The intensity difference of the different fluorophores reveals abundance differences.
- 'SHOTGUN' PROTEOMICS STRATEGY
-
A proteomics strategy that is based on the proteolysis of proteins, in particular, protein mixtures. The resulting peptide mixtures are usually analysed by liquid-chromatography–tandem mass spectrometry (LC–MS/MS) or multidimensional protein-identification technology (MudPIT).
- LEIGH SYNDROME
-
An early-onset progressive neurodegenerative disorder with a characteristic neuropathology that consists of focal, bilateral lesions in one or more areas of the central nervous system.
- PLASMALOGEN
-
Any glycerophospholipid in which a fatty acid group is replaced by a fatty aldehyde group.
- MALDI MS
-
(matrix-assisted laser desorption/ionization mass spectrometry). MALDI is a technique that uses a laser to desorb and ionize molecules that are co-crystallized into an organic matrix that absorbs strongly at the wavelength of the laser. MALDI MS is carried out using a mass spectrometer that uses laser-based ionization to create ions. MALDI is most often combined with a time-of-flight mass spectrometer.
- BREFELDIN A
-
An inhibitor of intracellular protein transport, which prevents GTP binding to the nucleotide-exchange factor ARF (ADP-ribosylation factor) and thereby inhibits protein transport through the Golgi apparatus.
- NYCODENZ
-
The trade name for a non-ionic iodinated chemical that is used to create density gradients.
- KDEL RECEPTOR
-
A Golgi-located receptor for the C-terminal KDEL sequence that is present on endoplasmic-reticulum-resident lumenal proteins.
- EDMAN SEQUENCING
-
A technique in which the N-terminal amino acids of a peptide are sequentially removed and identified in order to determine its amino-acid sequence.
- TRANSIT PEPTIDES
-
Protein localization to chloroplasts is mediated by cleavable N-terminal transit peptides, which are sequences of 30–100 amino acids that direct proteins to the chloroplast for translocation across chloroplast membranes.
- CALVIN CYCLE
-
The Calvin cycle is a metabolic pathway that occurs in the stroma of chloroplasts, in which carbon enters in the form of CO2 and leaves in the form of sugar. The cycle uses ATP as an energy source and NADPH as a reducing agent.
- SUBTRACTION STRATEGY
-
A process to subtract proteins that are found in a control state from an experimental or organelle-enriched state.
- MICROSOMAL MEMBRANE FRACTION
-
An enriched fraction of microsomal membranes, which includes the endoplasmic reticulum, mitochondria and ribosomes.
- DYSTROPHY
-
A degenerative disorder that mostly affects muscles.
- DIGITONIN SHIFT
-
Digitonin is used to perturb the density of membraneous compartments that contain cholesterol.When it is added in molar excess to cholesterol in membrane fractions, the density of the membranes is shifted in proportion to the cholesterol content, and the largest shift is seen for plasma membranes.
- LIPID RAFT
-
A type of lipid microdomain that is characterized by a relatively high content of cholesterol, sphingomyelin and glycosphingolipids. These microdomains are resistant to solubilization by non-ionic detergent because of their tight packing. They resemble liquid-ordered domains that are found in model membranes, which are characterized by tight packing and the high lateral mobility of lipids within the bilayer.
- SILAC
-
(stable-isotope labelling by amino acids in cell culture). Stable-isotope-labelled amino acids are added to a cell-culture media in order to label proteins.
- CENTROSOME
-
A structure that occurs close to the nucleus in eukaryotic cells during interphase. It comprises a pair of centrioles, satellite bodies and a cytoplasmic zone and, in animal cells, it serves as the main microtubule-organizing centre.
- 14-3-3 COMPLEXES
-
Discovered and named on the basis of their coordinates on two-dimensional gels, these cytosolic proteins bind to specific target proteins in a manner that is often regulated by the phosphorylation of their targets.
- FLAG TAGGED
-
A peptide-sequence epitope from bacteriophage T7 is fused to a protein to allow it to be recognized by a monoclonal antibody directed towards the epitope. This enables protein purification and localization.
- INTERPHASE
-
The period of the cell cycle between two mitotic divisions.
- SPINDLE POLES
-
The region of the mitotic spindle where microtubule minus ends are focused.
- PROTEIN-CORRELATION PROFILING
-
A consensus fractionation profile for peptides that can be assigned to known organelle proteins is established, and peptides that deviate from this profile are considered nonspecific. This technique allows the analysis of any organelle or cellular structure that can be enriched but not purified to homogeneity.
- KINETOCHORE
-
A multisubunit protein complex that is located at the pericentric region of DNA and that provides an attachment point for the spindle microtubules.
- CYTOKINESIS
-
The process by which a mother cell divides to form two daughter cells.
- MIDBODY
-
A structure that forms before a mother cell has completely divided to form two daughter cells. It is a dense proteinaceous structure that is associated with microtubules and is found at the site of division in animal cells.
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Yates III, J., Gilchrist, A., Howell, K. et al. Proteomics of organelles and large cellular structures. Nat Rev Mol Cell Biol 6, 702–714 (2005). https://doi.org/10.1038/nrm1711
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DOI: https://doi.org/10.1038/nrm1711
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