Stable Isotope Labeling with Amino Acids in Cell Culture. Thermo Scientific Pierce SILAC Protein Quantitation Kits

Transcription

1 Stable Isotope Labeling with Amino Acids in Cell Culture Thermo Scientific Pierce SILAC Protein Quantitation Kits

2 Quantitative Analysis of Differential Protein Expression Stable isotope labeling using amino acids in cell culture (SILAC) is a powerful method to identify and quantify relative differential changes in complex protein samples. SILAC uses in vivo metabolic incorporation of heavy C- or 15 N-labeled amino acids into proteins followed by mass spectrometry (MS)-based analysis, which accelerates the comprehensive identification, characterization and quantification of proteins. SILAC Technology 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 Identifying and quantifying hundreds to thousands of proteins in a single experiment The method requires growing mammalian cells in specialized media deficient in lysine and arginine amino acids. This deficiency is compensated by adding light or heavy forms of the missing amino acids; i.e., 12 C 6 and C 6 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. If a chemical treatment or a genetic manipulation is introduced to the cell population containing heavy amino acids, biological effects of the treatment or manipulation can be detected. The detection is made possible because the heavy and light amino acids are chemically identical to each other, yet isotopically distinct. This, in turn, makes the detection of the differentially expressed proteins possible based on relative peak intensity of the isotopic peptide pairs affected (Figure 1).

3 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 mammalian cell lines adapted to grow in DMEM or RPMI-1640, including HeLa, 293T, COS7, U2OS, A549, A431, HepG2, NIH 3T3, Jurkat and others Cells Grown in Light Isotope-containing Media Cells Grown in Heavy Isotope-containing Media + Treatment Harvest Cells Mix Cell Lysates Excise Bands 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 provide excellent data for SILAC studies and general proteomics, metabolomics and small molecule research applications. For more information, visit SDS-PAGE Trypsin Digestion LC-MS/MS Ratio Determination m/z Heavy Figure 1. Schematic of SILAC workflow. A549 cells adapted to DMEM were grown for six passages (10 days) using SILAC DMEM (Thermo Fisher Scientific, Rockford, IL, Product # 89983) containing 0.1 mg/ml heavy C 6 L-lysine-2HCl or light L-lysine-HCl supplemented with 10% dialyzed FBS. After 100% label incorporation, C 6 L-lysine-labeled cells were treated with 5 μm camptothecin (Sigma, St. Louis, Product # C9911) for 24 hours. Cells from each sample (light and heavy) were lysed using Thermo Scientific M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Rockford, IL, Product # 78501). Samples were normalized for protein concentration using the Thermo Scientific Pierce BCA Protein Assay (Thermo Scientific, Rockford, IL, Product # 23225), and 50 μg of each sample were equally mixed before 4-20% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis. Gels were stained with Thermo Scientific GelCode Blue Stain Reagent (Thermo Fisher Scientific, Rockford, IL, Product # 24592) and proteins were digested and alkylated using the Thermo Scientific Pierce In-Gel Tryptic Digestion Kit (Thermo Fisher Scientific, Rockford, IL, Product # 89871) before analysis using an LTQ Orbitrap Hybrid Mass Spectrometer. Relative Intensity Light

4 The Thermo Scientific Pierce SILAC Protein Quantitation 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 C 6 15 N 4 L-Arginine is available separately (Product # 89990) and can be combined with 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 lowabundance proteins; cell-surface, organelle- specific proteins; and post-translational modifications such as phosphorylation or glycosylation. Protein levels from A549 lung carcinoma cells treated with the anti-cancer drug camptothecin (Figure 1) were analyzed by MS. Most of the proteins identified had no change in abundance level after camptothecin treatment; however, % of proteins quantified in heavy-labeled cells had protein levels (SILAC ratios) 1.5-fold higher than control cells (Figure 2). More than 6% of proteins had their relative abundance reduced by the same amount after drug treatment. Fifteen proteins were identified by MS as the most significantly upregulated after drug treatment (Table 1). One protein that was identified as being upregulated in response to camptothecin treatment was proliferating cell nuclear antigen (PCNA), a protein known to be involved in DNA repair (Figure 3). 10 H:L α - PCNA 1.9 SILAC Ratio α - p α - β-actin 1.2 Relative Abundance 0.1 Protein Number Figure 2: SILAC ratio distribution. Comparison of A549 protein levels by SILAC ratio after camptothecin treatment. MS/MS analysis of peptides generated (Figure 1) identified 450 proteins of which 361 contained C 6 L-lysine. SILAC ratios were calculated using Thermo Scientific BioWorks Software with PepQuan SILAC quantitation with the following settings: target ions = residue = K, mass = 6.0, mass tolerance = 0.1, minimum threshold = 10, no smoothing, and perform calculation base on area. SILAC ratios for all 361 proteins are plotted on a logarithmic graph SILAC Ratio H:L = Light (L) m/z Heavy (H) Figure 3: Representative MS spectra generated using SILAC. Light and heavy ( C 6 ) L-lysine-containing peptide (AEDNADT- LALVFEAPNQEK) from PCNA were analyzed by MS. Mass spectra of heavy peptides containing C 6 L-lysine, which have an increase mass of 6 Da, seen here as a shift of 3 m/z, given that this is a +2 charge state peptide. The SILAC ratio was determined by comparing relative abundance of heavy (H) and light (L) peptides (Figure 2). Light (L) Heavy (H) - + α - GAPDH 5 μm camptothecin Figure 4: Comparison of A549 protein levels detected by Western blotting after camptothecin treatment. Ten micrograms of each light (L) and heavy (H) sample (Figure 1) was analyzed by 4-20% SDS-PAGE and Western blotting using specific Thermo Scientific Lab Vision Antibodies against PCNA (Thermo Fisher Scientific, Fremont, CA, Product # MS-106-PO) diluted to 1:1,000 or Thermo Scientific Lab Vision p53 (Thermo Fisher Scientific, Fremont, CA, Product # MS-186-PI), b-actin (US Biological, Swampscott, MA, Product # AO760-40) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, Product # SC-25778) diluted 1:10,000. Anti-Mouse (H+L)-HRP (Thermo Fisher Scientific, Rockford, IL, Product # 31430) or Anti-Rabbit (H+L)-HRP (Thermo Fisher Scientific, Rockford, IL, Product # 31460) diluted 1:25,000 were used as the secondary antibody with SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific, Rockford, IL, Product # 34076) for detection. Protein levels (H:L) were quantitated after Western blotting using a Kodak Image Station 2000MM. 1.1

5 To validate SILAC data, protein levels were separately quantitated by Western blot (Figure 4). The proteins levels increased 1.9- and 5.9-fold for PCNA and p53, respectively. However, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin did not significantly change. The abundance ratios determined by Western blot of PCNA and GAPDH were comparable to those determined by SILAC (Table 1). The p53 protein was not identified by MS due to its relative low abundance in cell extracts. Table 1: The 15 most up-regulated proteins identified in heavylabeled A549 whole cell lysates after camptothecin treatment. SILAC Ratio H:L Protein* (# Peptides) Isoform Short of NADPH:adrenodoxin 4.50 (3) oxidoreductase, mitochondrial precursor Plastin (2) TUBB6 protein 2.35 (2) Superoxide dismutase [Mn], mitochondrial 2.24 (2) precursor Isoform M1 of Pyruvate kinase isozymes M1/M (2) Actin, cytoplasmic (12) Transgelin (2) UDP-glucose ceramide glucosyltransferase-like 2.11 (3) 1 isoform 1 Proliferating cell nuclear antigen (PCNA) 2.09 (3) Isoform 1 of Spectrin alpha chain, brain 1.96 (2) Isoform 2 of Nucleophosmin 1.93 (3) Vimentin 1.92 (12) Isoform 1 of Clathrin heavy chain (7) Actin, alpha skeletal muscle 1.86 (2) Keratin, type II cytoskeletal (8) Glyceraldehyde-3-phosphate dehydrogenase 1.09 (3) (GAPDH) * Proteins were identified by the SEQEUST program using the International Protein Index database (IPI:Human, V3.18). Ordering Information: Product # Description Pkg. Size Pierce SILAC Protein Kit Quantitation Kit RPMI 1640 Includes: SILAC RPMI Media 2 x 500 ml Dialyzed FBS 2 x 50 ml C 6 L-Lysine-2HCl 50 mg L-Lysine-2HCl 50 mg L-Arginine-HCl 2 x 50 mg Pierce SILAC Protein Kit Quantitation Kit DMEM Includes: SILAC DMEM media 2 x 500 ml Dialyzed FBS 2 x 50 ml C 6 L-Lysine-2HCl 50 mg L-Lysine-2HCl 50 mg L-Arginine-HCl 2 x 50 mg SILAC RPMI Media 500 ml (RPMI-1640 Medium minus L-Lysine and L-Arginine) SILAC DMEM Media 500 ml (DMEM Medium minus L-Lysine and L-Arginine) Dialyzed FBS 50 ml L-Lysine-2HCl 50 mg C6 L-Lysine-2HCl 50 mg L-Arginine-HCl 50 mg C6 15N4 L-Arginine-HCl 50 mg Complementary Products: Product # Description Pkg. Size Halt Protease Inhibitor Cocktail Kit Halt Phosphatase Inhibitor Cocktail 1 ml M-PER Mammalian Protein 250 ml Extraction Reagent Mem-PER Membrane Protein Kit Extraction Reagent NE-PER Nuclear and Cytoplasmic Kit Extraction Reagents Mitochondria Isolation Kit Kit Lysosome Enrichment Kit Kit for Tissue and Cultured Cells Peroxisome Enrichment Kit Kit for Tissue Nuclei Enrichment Kit for Tissue Kit For information on Pierce Protein Research Products, visit For information on LTQ Orbitrap Hybrid Mass Spectrometers, visit For information on Cell Culture and Bioprocessing Systems, visit

6 /07 Pierce Protein Research Products Tel: or Fax: Customer Assistance Outside the United States, visit our website or call to locate your local branch office or distributor Thermo Fisher Scientific Inc. All rights reserved. These products are supplied for laboratory or manufacturing applications only. Prices listed herein were accurate at the time of printing. Visit our website for up-to-date prices. SuperSignal Technology is protected by U.S. Patent # 6,432,662. Pierce BCA Technology is protected by U.S. Patent # 4,839,295. Kodak is a trademark of Eastman Kodak Corporation. All other trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries.