DMEM and RPMI kits and reagents for stable isotope labeling using amino acids in cell culture (SILAC).

Stable isotope labeling with amino acids in cell culture (SILAC) is a powerful method to identify and quantify relative differential changes in complex protein samples. The SILAC Method uses in vivo metabolic incorporation of "heavy" ¹³C- or ¹⁵N-labeled amino acids into proteins followed by mass spectrometry (MS) analysis for accelerated comprehensive identification, characterization and quantitation of proteins.

SILAC Highlights:

• Efficient – 100% label incorporation into proteins of living cells
• Reproducible – eliminates intra-experimental variability caused by differential sample preparation
• Flexible – media deficient in both L - lysine and L - arginine, allowing for more complete proteome coverage through dual amino acid isotope labeling
• Compatible – label proteins expressed in a wide variety of mammalian cell lines adapted to grow in DMEM or RPMI 1640 medium, including HeLa, 293T, COS7, U2OS, A549, A431, HepG2, NIH 3T3, Jurkat and others

SILAC Applications:

• Quantitative analysis of relative changes in protein abundance from different cell treatments
• Quantitative analysis of proteins for which there are no antibodies available
• Protein expression profiling of normal vs. disease cells
• Identification and quantification of hundreds to thousands of proteins in a single experiment

Figure 1. Example SILAC workflow. A549 cells adapted to DMEM were grown for six passages (10 days) using SILAC DMEM containing 0.1 mg/ml heavy 13C6 L-lysine-2HCl or light L-lysine-HCl supplemented with 10% dialyzed FBS. After 100% label incorporation, heavy-labeled cells were treated with 5 µm camptothecin for 24 hours. Cells from each sample (light and heavy) were lysed using Thermo Scientific M-PER Reagent (Product # 78501). Samples were normalized for protein concentration using the Thermo Scientific Pierce BCA Protein Assay (Product # 23225), and 50 µg of each sample were equally mixed before 4-20% SDS-PAGE. Gels were stained with Thermo Scientific GelCode Blue Stain Reagent (Product # 24592) and proteins were digested and alkylated using the Thermo Scientific Pierce In-Gel Tryptic Digestion Kit (Product # 89871) before analysis using an LTQ Orbitrap Hybrid Mass Spectrometer.

The stable isotope labeling of amino acids in cell culture method requires growing mammalian cells in specialized media deficient in lysine and arginine. This deficiency is compensated by adding light or heavy forms of the missing amino acids; i.e., ¹²C₆ and ¹³C₆ L-lysine, respectively. A typical experiment involves growing one cell population in medium containing light amino acids (control), while the other population is grown in the presence of heavy amino acids (experimental). The heavy and light amino acids are incorporated into proteins through natural cellular protein synthesis. After alteration of the proteome in one sample through chemical treatment or genetic manipulation, equal amounts of protein from both cell populations are then combined, separated by SDS polyacrylamide gel electrophoresis and digested with trypsin before MS analysis. Because peptides labeled with heavy and light amino acids are chemically identical, they co-elute during reverse-phase column pre-factionation and, therefore, are detected simultaneously during MS analysis. The relative peak intensities of multiple isotopically distinct peptides from each protein are then used to determine the average change in protein abundance in the treated sample (Figure 1).

Kits are compatible with mammalian cell lines adapted to grow in either DMEM or RPMI 1640 media. Each kit includes all necessary reagents to isotopically label cells, including media, heavy and light amino acids, and dialyzed serum. The heavy ¹³C₆ ¹⁵N₄ L-Arginine is available separately and can be combined with Pierce SILAC Protein Quantitation Kits to enhance peptide isotope label coverage. When combined with Thermo Scientific Protein/Peptide Sample Enrichment Products, Pierce SILAC Protein Quantitation Kits also enable MS analysis of low-abundance proteins such as cell-surface proteins, organelle-specific proteins and protein post-translational modifications such as phosphorylation or glycosylation.

Data
Using a Pierce SILAC Quantitation Kit, A549 cells adapted to grow in Dulbecco's Modified Eagle medium (DMEM) were labeled with ¹³C₆ L-lysine to > 98% isotope incorporation. Heavy-labeled cells treated with camptothecin were lysed, mixed with control lysates, separated by SDS-PAGE and digested with trypsin before MS analysis. More than 350 proteins were successfully identified by MS/MS sequencing using a Thermo Scientific Finnigan LTQ Orbitrap Mass Spectrometer. Identified peptides were then quantitated using the Thermo Scientific Bioworks Software Suite to generate SILAC ratios corresponding to relative changes in protein abundance.

Most of the proteins identified had no change in abundance level after camptothecin treatment (Figure 2); however, 13% of proteins quantified in heavy-labeled cells had protein levels (SILAC ratios) 1.5-fold higher than control cells. More than 6% of proteins had their relative abundance reduced by the same amount after drug treatment while 15 proteins were identified by MS as the most significantly up-regulated after drug treatment (Table 1). One protein that was identified as being up-regulated in response to camptothecin treatment was proliferating cell nuclear antigen (PCNA), a protein known to be involved in DNA repair (Figure 3). To validate SILAC data, protein levels were separately quantitated by Western blot (Figure 4). PCNA protein levels increased 1.9-fold; however, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping protein did not significantly change. The abundance ratios determined by Western blot were comparable to those determined by SILAC (Table 1).

The Thermo Scientific LTQ Orbitrap Hybrid Mass Spectrometer enables faster and more reliable detection and identification of proteins in complex mixtures. Routine high-resolution and mass measurement accuracy of 2-3 ppm provides excellent data for SILAC studies and general proteomics, metabolomics, and small molecule research applications. For more information, visit www.thermo.com/orbitrap.

The following Poster Presentations were presented at the American Society for Mass Spectrometry, June 2007:

• Quantitation of Chemical and Genetic Alteration of the Proteome using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)
• Protein Quantitation of p53-Regulated Proteins using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)
• Using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) to Quantitate Exportin/CRM1-dependent Protein Transport in Nuclear Extracts

SILAC Literature References

1. Amanchy, R. et al. (2005). Science STKE 267: 1-20
2. Blagoev, B., et al. (2004). Nat Biotechnol 22(9): 1139-1145.
3. Everley, P. et al. as presented at The American Society for Biocehmistry and Molecular Biology. MCP Papers in Press. Published on July 11, 2007 as Manuscript M700057-MCP200.
4. Kratchmarova, I., et al. (2005). Science 308(5727): 1472-1477.
5. Mann, M. (2006). Nat Rev Mol Cell Biol 7(12): 952-958.
6. Ong, S. E., et al. (2002). Mol Cell Proteomics 1(5): 376-386.
7. Selbach, M. and Mann, M. (2006). Nat. Methods 3(12): 981-3

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AQUA and Heavy Peptides – custom peptide isotopes for mass spectrometry
SILAC Protein Quantitation Kits FAQ
SILAC Flier
Prepare SILAC peptides using the In-Gel Tryptic Digestion Kit (Tech Tip #60)

The purchase of this product conveys a non-transferable license to the Purchaser to use this product in methods protected under U.S Patent 6,653,076 (owned by University of Washington) for research purposes only.