Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
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.
Protein Interaction Laboratory, University of Southern Denmark, Odense, Denmark.
Shao-En
SE
Ong
Blagoy
B
Blagoev
Irina
I
Kratchmarova
Dan Bach
DB
Kristensen
Hanno
H
Steen
Akhilesh
A
Pandey
Matthias
M
Mann
D016475:3T3 Cells; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D003903:Deuterium / Q000378:metabolism; D005821:Genetic Techniques; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000378:metabolism; D051379:Mice; D009132:Muscles / Q000166:cytology; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D013997:Time Factors; D015854:Up-Regulation
D000596:Amino Acids; D010455:Peptides; D003903:Deuterium; D007930:Leucine