Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol 7:952-958

Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Nature Reviews Molecular Cell Biology (Impact Factor: 37.81). 01/2007; 7(12):952-8. DOI: 10.1038/nrm2067
Source: PubMed


Researchers in many biological areas now routinely characterize proteins by mass spectrometry. Among the many formats for quantitative proteomics, stable-isotope labelling by amino acids in cell culture (SILAC) has emerged as a simple and powerful one. SILAC removes false positives in protein-interaction studies, reveals large-scale kinetics of proteomes and - as a quantitative phosphoproteomics technology - directly uncovers important points in the signalling pathways that control cellular decisions.

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    • "We selected one of the more promiscuous compounds, JJH221 (4), for analysis by mass spectrometry (MS)-based ABPP methods to assess the range of SHs targeted by NHH carbamates . JJH221-sensitive SHs were identified using the quantitative MS method ABPP-SILAC (stable isotope labeling by amino acids in cell culture; Mann, 2006) (Adibekian et al., 2011). In brief, proteomes from isotopically heavy-and lightamino acid-labeled human PC3 cells were treated with JJH221 (20 mM) or DMSO, respectively, for 4 hr followed by the biotinylated FP probe FP-biotin (Liu et al., 1999) (2.5 mM, 1 hr). "
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    ABSTRACT: Serine hydrolase inhibitors, which facilitate enzyme function assignment and are used to treat a range of human disorders, often act by an irreversible mechanism that involves covalent modification of the serine hydrolase catalytic nucleophile. The portion of mammalian serine hydrolases for which selective inhibitors have been developed, however, remains small. Here, we show that N-hydroxyhydantoin (NHH) carbamates are a versatile class of irreversible serine hydrolase inhibitors that can be modified on both the staying (carbamylating) and leaving (NHH) groups to optimize potency and selectivity. Synthesis of a small library of NHH carbamates and screening by competitive activity-based protein profiling furnished selective, in vivo-active inhibitors and tailored activity-based probes for multiple mammalian serine hydrolases, including palmitoyl protein thioesterase 1, mutations of which cause the human disease infantile neuronal ceroid lipofuscinosis. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Chemistry & biology 06/2015; 22(7). DOI:10.1016/j.chembiol.2015.05.018 · 6.65 Impact Factor
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    • "In SILAC, two populations of cells are grown in two different culture media, with the " light " medium containing amino acid(s) with the natural isotope, and the " heavy " medium containing stable isotope labeled amino acid(s). After a sufficient number of cell divisions, at least five cycles in mammalian cells [21], theoretically all the proteins from the cells cultured in heavy medium contain amino acids in the heavy state. However, the number of cell divisions required for complete labeling depends on the rate of protein synthesis, degradation, and turnover, therefore the labeling efficiency should be carefully tested prior to quantification . "
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    ABSTRACT: Stable isotope labeling by amino acids in cell culture (SILAC) is based on direct addition of selected stable isotope amino acids into the cell culture medium, allowing superior quantitative analysis of the cellular proteome compared to other labeling methods. The great advantages of SILAC lie in its straight-forward implementation, quantitative accuracy and reproducibility over chemical labeling or label-free quantification strategies, favoring its adoption for proteomic research. SILAC has been widely applied to characterize the proteomic changes between different biological samples, to investigate dynamic changes of protein post-translational modifications (PTMs), to distinguish specific interacting proteins in interaction proteomic analysis, and to analyze protein turnover in the proteome-wide scale. The present review summarizes the principles of SILAC technology, its applications in biological research, and the present state of this technology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Proteomics 06/2015; 15(18). DOI:10.1002/pmic.201500108 · 3.81 Impact Factor
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    • "The most commonly used quantitative strategy in proteomics is relative quantitation. This can be achieved using several different techniques, for example the stable isotope labeling by amino acids in cell culture (SILAC) approach [55] [56], various chemical labeling procedures, e.g. trypsin-catalyzed 18 O labeling [57] or dimethyl labeling [58] [59] or by isobaric labeling, i.e. "
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    ABSTRACT: Post-translational modifications (PTM) of proteins determine the activity, stability, specificity, transportability and lifespan of a protein. Some PTM are highly specific and regulated involving various enzymatic pathways, but there are other non-enzymatic PTM (nePTM), which occur stochastically, depend on the ternary structure of proteins and can be damaging. It is often observed that inactive and abnormal proteins accumulate in old cells and tissues. The nature, site and extent of nePTM give rise to a population of that specific protein with alterations in structure and function ranging from being fully active to totally inactive molecules. Determination of the type and the amount (abundance) of nePTM is essential for establishing connection between specific protein structure and specific biological role. This article summarizes analytical demands for reliable quantification of nePTM, including requirements for the assay performance, standardization and quality control, and points to the difficulties, uncertainties and un-resolved issues. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
    04/2015; 5. DOI:10.1016/j.redox.2015.04.001
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