Key Points
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Mass spectrometry (MS) has become a powerful and indispensable tool to study different aspects of cell signalling, such as the identification and quantification of post-translational modifications (PTMs), characterization of protein–protein interactions and analysis of changes in protein expression.
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Dramatic advances in all aspects of the proteomic workflow, and especially in computational proteomics, have enabled the quantification of a first complete proteome.
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Stable isotope labelling by amino acids in cell culture (SILAC) and isobaric tag for relative and absolute quantification (iTRAQ) are most widely used for MS-based quantification of proteins and PTMs. The scope for label-free quantification algorithms is likely to grow, especially in cases where expected changes in proteins or PTMs are large and accurate quantification is not essential.
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High resolution quantitative MS has been successfully applied to quantify thousands of phosphorylation and acetylation sites. These data provide a basis for directed functional analysis on single proteins as well as for 'systems-wide' studies.
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There are now pioneering examples of the application of high resolution quantitative MS in elucidating global signalling networks and their dynamics in response to different cellular perturbations.
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Bioinformatic analysis of proteomic data can provide insights into the nature and evolution of signalling networks and global kinase–substrate relationships.
Abstract
Signalling networks regulate essentially all of the biology of cells and organisms in normal and disease states. Signalling is often studied using antibody-based techniques such as western blots. Large-scale 'precision proteomics' based on mass spectrometry now enables the system-wide characterization of signalling events at the levels of post-translational modifications, protein–protein interactions and changes in protein expression. This technology delivers accurate and unbiased information about the quantitative changes of thousands of proteins and their modifications in response to any perturbation. Current studies focus on phosphorylation, but acetylation, methylation, glycosylation and ubiquitylation are also becoming amenable to investigation. Large-scale proteomics-based signalling research will fundamentally change our understanding of signalling networks.
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Acknowledgements
We thank our co-workers for fruitful discussions, especially J. Olsen for providing figure 3. The Protein Research Center (CPR) is supported by a generous donation from the Novo Nordisk Foundation. This project was supported by the European Commission's 7th Framework Program PROteomics SPECification in Time and Space (PROSPECTS, HEALTH-F4-2008-021,648).
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Glossary
- SH2 domain
-
(SRC homology 2 domain). An ∼100 amino acid domain that recognizes phosphoTyr residues in a specific sequence context.
- PTB domain
-
(PhosphoTyr-binding domain). Like the SH2 domain, the PTB domain binds to phosphoTyr, but usually binding specificity is determined by the sequence N-terminal to the phosphorylation site.
- Liquid chromatography
-
In high performance liquid chromatography (HPLC), the peptide mixture is separated in liquid phase based on hydrophobic interactions with the C18 stationary phase of the chromatography column (C18 is the length of the alky chains decorating the chromatographic beads). In proteomics, columns are typically very small (75 m inner diameter) and flow rates very low (200 nl min−1).
- Electrospray ionization
-
An ionization method developed by J. Fenn, for which he shared the 2002 Nobel Prize in chemistry. A liquid is passed through a charged needle, producing electrosprayed droplets that contain the peptides. On evaporation of the solvent, intact and protonated peptides (or other analyte molecules) are left in the gas phase.
- Mass analyser
-
A part of a mass spectrometer that measures mass to charge (m/z) ratios of ions (for example, ionized peptides). Multiplying the m/z value by the charge and subtracting the weight of the charging entity (typically two protons) yields the mass of the peptide. A mass spectrometer can contain several mass analysers of the same or different types, and ions can be moved between these analysers at will.
- Fourier transformation
-
A mathematical operation that transforms one complex-valued function of a real variable (typically a frequency spectrum) into another domain. In Fourier transformation MS, the frequencies associated with ions moving in a trap are mass dependent and this signal is transformed by Fourier transformation into a mass spectrum.
- MS resolution
-
This value is defined as the width of the peak at half height divided by the mass of the peak and is therefore a dimensionless number. High resolution distinguishes co-eluting peptides with similar mass, a prerquisite for unambiguous identification and quantification of peptides.
- Chemical derivatization
-
A chemical method used to transform one chemical compound into a derivative. In proteomics, side chains of amino acids can be chemically modified, which can be used for enriching these peptides from complex mixtures or for quantification of the modified peptide in MS.
- False discovery rate
-
(FDR). A statistical method used in multiple hypotheses testing to correct for multiple comparisons. In a list of positive calls, FDR controls the expected proportion of false positives. In proteomic data analysis, a 1% FDR is currently customary, which means that, at most, 1% of the identified proteins should be false positives.
- Metal affinity complexation
-
The coordinated binding between immobilized metal ions and charged peptides. Immobilized metals such as Fe3+ or Ga3+, or metal oxides such as TiO2 or ZrO2, are commonly used to enrich phosphorylated peptides from non-phosphorylated peptides.
- F-actin cup
-
(Filamentous actin cup). A polymer of globular actin (G-actin) subunits, which accumulates underneath phagocytic cups — cup-shaped extensions of the plasma membrane that encircle foreign particles during early processes of phagocytosis.
- Endocytic route
-
The trafficking of cell surface receptors from the plasma membrane to intracellular compartments by receptor endocytosis, which generally involves early and late endosomes, multivesicular bodies and lysosomes. It is a major pathway that regulates the amplitude and duration of receptor signalling at the plasma membrane.
- Biosynthetic route
-
The trafficking of receptors from their intracellular biosynthesis compartments to the surface, involving protein synthesis at the ER, relocation to the Golgi and, finally, transport to the cell surface.
- FLT3-ITD
-
The FLT3 receptor containing an in-frame insertion of amino acid sequence (internal tandem duplication (ITD); of ∼3–100 amino acids in length) in the intracellular juxtamembrane region, which results in ligand-independent receptor activation. These oncogenic mutations are found in about 20% of human acute myeloid leukaemia and are associated with oncogenic transformation.
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Choudhary, C., Mann, M. Decoding signalling networks by mass spectrometry-based proteomics. Nat Rev Mol Cell Biol 11, 427–439 (2010). https://doi.org/10.1038/nrm2900
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DOI: https://doi.org/10.1038/nrm2900
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