Introduction of Phosphorylation
Phosphorylation, a widely observed posttranslational modification, holds significant importance in the realm of protein regulation, particularly within the complex machinery of eukaryotic cells. Within these cellular systems, phosphorylation predominantly targets specific amino acid residues such as serine, threonine, and tyrosine. While serine and threonine phosphorylation occur more frequently, tyrosine phosphorylation stands out due to its pivotal role in orchestrating intracellular signal transduction processes.
In intricate signaling cascades such as receptor tyrosine kinase (RTK) pathways, the initiation of signaling events is often triggered by the binding of extracellular ligands to their corresponding receptors. This event catalyzes a cascade of molecular interactions, ultimately leading to the phosphorylation of tyrosine residues within the receptor itself, as well as on downstream effector proteins. This phosphorylation acts as a molecular switch, activating or deactivating various cellular pathways and ultimately regulating diverse cellular processes.
Why Choose SILAC?
Conventional methodologies employed in the study of these intricate signaling pathways often face limitations in providing comprehensive insights. However, the advent of mass spectrometry (MS)-based quantitative proteomics has revolutionized this field, offering a robust platform for the high-throughput investigation of cellular signal transduction processes. Among the various techniques, Stable Isotope Labeling by Amino acids in Cell culture (SILAC) has emerged as an exceptionally effective approach for achieving precise and accurate quantitative proteomics data.
SILAC methodology boasts several advantages, including its user-friendly nature and the high level of incorporation of labeled amino acids into proteins. One of its notable features is its versatility, as SILAC is not confined to mammalian cells but can be successfully applied across a wide range of organisms. This versatility enhances its utility in diverse biological contexts.
When coupled with anti-phosphotyrosine (pTyr) immunoprecipitation (IP), SILAC becomes a potent tool specifically tailored for the study of receptor tyrosine kinase (RTK) signaling pathways. This integrated approach enables the identification and quantification of tyrosine-phosphorylated proteins, along with their associated binding partners, providing valuable insights into the dynamics of RTK signaling networks.
Material for Phosphotyrosine Protein Identification by SILAC
Cell Culture and SILAC Labeling:
- Cell line: NG108-15 cell line, a hybrid of mouse neuroblastoma and rat glioma stably over-expressing EphB2.
- Culture medium: Dulbecco's Modified Eagle Medium (DMEM) deficient in lysine and arginine.
- Amino acids for labeling: Normal (light) L-lysine and L-arginine hydrochloride, and stable isotope-labeled (heavy) L-lysine 13C6 and L-arginine 13C6 hydrochloride.
- Supplements: Dialyzed fetal bovine serum, post-fusion selective medium HAT, penicillin/streptomycin, plasmid selecting antibiotic G418.
- Other: Sterile phosphate buffered saline (PBS), trypsin-EDTA solution, 0.22-μm filter flasks.
- Ligand stimulation: EphrinB1-Fc, recombinant human Fc, and goat anti-human Fc IgG.
Lysis buffer: Contains Triton X-100, NaCl, Tris–HCl (pH 8.0), EDTA, tyrosine phosphatase inhibitor sodium orthovanadate, serine/threonine phosphatase inhibitor sodium fluoride, and protease inhibitors.
Agarose conjugated anti-pTyr antibody PY99: Used for targeting phosphorylated tyrosine residues in proteins.
Elution buffer: Comprises trifluoroacetic acid (TFA) and SDS for releasing proteins during purification.
Precast 7.5% Tris–HCl polyacrylamide gel: Utilized for protein separation.
Coomassie brilliant blue R250 (CBB) staining solution: Staining solution for visualizing proteins after electrophoresis.
Destaining buffer: Contains ammonium bicarbonate and acetonitrile for removing excess stain from gels.
Acetonitrile (HPLC grade): High-purity solvent used in various steps of analysis.
Trypsin solution: Enzyme solution containing trypsin for digesting proteins into peptides.
Extraction buffer: Solution of formic acid and acetonitrile for protein extraction.
Procedure
SILAC Medium Preparation:
- Prepare stock solutions of light and heavy labeling amino acids by dissolving them in PBS.
- Dilute these solutions 1,000-fold with DMEM deficient in arginine and lysine.
- Supplement the media with HAT, penicillin/streptomycin, and G418 antibiotics.
- Filter the media to ensure sterility and add 10% dialyzed fetal bovine serum to support cell growth.
SILAC Cell Culture:
- Split NG108-EphB2 cells into two dishes: one for light medium and one for heavy medium.
- Allow the cells to grow and divide, ensuring they undergo at least five doublings to achieve complete labeling.
Cell Stimulation and Lysis:
- Starve the cells in serum-free SILAC media to synchronize their growth and lower basal tyrosine phosphorylation levels.
- Cluster the ligand by incubating ephrinB1-Fc and anti-human Fc, then add to the cells for stimulation.
- Wash the cells with cold PBS, lyse them with ice-cold lysis buffer, and centrifuge to collect the lysates.
- Mix equal amounts of light and heavy lysates for downstream analysis.
Anti-pTyr IP and SDS-PAGE:
- Pre-clear the lysates and perform immunoprecipitation with agarose-conjugated anti-phosphotyrosine antibody PY99.
- Wash the beads to remove non-specifically bound proteins and elute the precipitated proteins using elution buffer.
- Neutralize the eluate, concentrate it, and load onto a precast gel for SDS-PAGE analysis.
In-Gel Digestion:
- Cut the gel bands into small pieces and destain them with ammonium bicarbonate/acetonitrile solution.
- Rehydrate the gel pieces with trypsin solution and incubate overnight for digestion.
- Extract the peptides from the gel pieces, concentrate them, and reconstitute them for subsequent LC-MS analysis.
Reference
- Zhang, Guoan, and Thomas A. Neubert. "Use of stable isotope labeling by amino acids in cell culture (SILAC) for phosphotyrosine protein identification and quantitation." Phospho-Proteomics: Methods and Protocols (2009): 79-92.
