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Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics
Open accessAbstract
Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.
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“In order to accurately compare the TTR values between individual brain samples and across cohorts of animals, we developed standard curves for both apoE and Aβ using the stable isotope labeling of amino acids in cell culture (SILAC) method [35].”
From Measurement of apolipoprotein E and amyloid β clearance rates in the mouse brain using bolus stable isotope labeling (2012) · cites this paper as [35]
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“Metabolic labeling methods such as SILAC1 (38) excel because of their unparalleled accuracy and robustness, which are mainly due to stability with regard to variability in sample processing and analysis steps.”
From Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ (2014) · cites this paper as [38]
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Metadata
- Authors
- Ong, S.-E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., & Mann, M.
- Affiliations
- University of Southern Denmark (DK)
- Corresponding author
- Mann, Matthias
- ORCID
- Venue
- Molecular & Cellular Proteomics
- Publisher
- Elsevier BV
- Published
- 2002-05
- Volume
- 1
- Issue
- 5
- Pages
- 376-386
- ISSN
- 1535-9476 (ISSN-L)
- OpenAlex
- W2140946052
- Cited by
- 5,574
- References
- 41
- Citations by year
- 2026: 69 · 2025: 89 · 2024: 117 · 2023: 142 · 2022: 172 · 2021: 241 · 2020: 192 · 2019: 266 · 2018: 233 · 2017: 300 · 2016: 299 · 2015: 349 · 2014: 397 · 2013: 440 · 2012: 430
- FWCI
- 34.74
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular & Cellular Proteomics (publishedVersion, cc-by)
- PubMed (publishedVersion)
- University of Southern Denmark Research Portal (University of Southern Denmark) (submittedVersion)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Keywords
- SILAC, in vivo protein labeling, quantitative proteomics, protein expression profiling, muscle cell differentiation, fibronectin, PKM2, glyceraldehyde-3-phosphate dehydrogenase
- MeSH terms
- Amino Acids, Animals, Cell Differentiation, Cell Line, Deuterium, Genetic Techniques, Hydrogen-Ion Concentration, Leucine, Muscles, Peptides, Time Factors, Up-Regulation, 3T3 Cells, Cell Culture Techniques, Proteomics, Mice
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