Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is widely used in quantitative proteomics for metabolic labeling. Compared to chemical labeling methods such as iTRAQ and TMT, SILAC naturally avoids biases introduced during sample processing by completing isotope labeling at the cellular level, making it an important tool for studying post-translational modifications (PTMs), especiallyphosphorylation modifications. This article will explore how SILAC aids phosphorylation modification research, and introduce real cases on how to obtain more biologically meaningful results through high-resolution mass spectrometry platforms.
The basic concept of SILAC is to add 'light' amino acids (such as [^12C6]-lysine) and 'heavy' amino acids (such as [^13C6]-lysine) to the cell culture medium, allowing proteins to introduce stable isotopes during synthesis through multiple generations of culture, thus presenting characteristic mass differences in mass spectrometry. Cells from different treatment groups are mixed after SILAC labeling and processed uniformly for lysis, digestion, enrichment, and finally quantitative analysis of proteins or modified peptides using mass spectrometry.
II. Advantages of SILAC in Phosphorylation Modification Research
1. High-precision Quantitative Capability
Phosphorylation is a highly dynamic modification that responds rapidly to changes in signaling pathways. Since SILAC labels samples at the cellular level and all subsequent experimental steps are conducted on mixed samples, it effectively avoids batch effects, enhancing the accuracy and reproducibilityof quantification, making it highly suitable for capturing dynamic phosphorylation changes.
2. Strong Compatibility with Phosphopeptide Enrichment Techniques
SILAC samples can be directly combined with phosphopeptide enrichment strategies such as TiO₂, IMAC, and Fe-NTA, achievingspecific enrichment and quantification of low-abundance phosphopeptides. This is particularly crucial for studying key regulatory sites in signaling pathways.
3. Enables Time-resolved Kinetic Analysis
By using triple labeling (such as light, medium, heavy), SILAC can simulate different time points after signal stimulation, trackingthe temporal dynamic changes of phosphorylation modifications, revealing the mechanisms of signal pathway initiation, amplification, and termination.
III. Application Scenarios of SILAC in Phosphorylation Modification Research
1. Analyzing Kinase-substrate Networks
For example, in researching signal transduction under EGF (epidermal growth factor) stimulation, SILAC can be used to label treatment and control group cells separately, enrich phosphopeptides for mass spectrometry analysis, thereby identifying downstream phosphorylation targets of the EGFR kinase and mapping kinase-substrate regulatory networks.
2. Research on Drug Mechanisms
For the development of targeted kinase inhibitors, SILAC can be used to monitor overall changes in phosphorylation sites in cells before and after drug treatment, assessing the regulatory effects of drugs on signaling pathways and potential off-target risks.
3. Cancer Signaling Pathway Research
Cancer cells are often accompanied by abnormal activation of phosphorylation signals. SILAC combined with phosphopeptide enrichment and high-resolution mass spectrometry can systematically compare phosphorylation differences between tumor and normal cells, providing data support for target discovery.
IV. Overview of Experimental Procedures for SILAC Phosphorylation Modification Research
1. Cell Culture: Cultivate for 3~6 generations using medium containing light/heavy labeled amino acids
2. Intervention Treatment: Such as drug stimulation, kinase activation or inhibition
3. Mixing Samples: Mix cell lysates proportionally under different conditions
4. Protein Digestion: Digest proteins into peptides using trypsin
5. Phosphopeptide Enrichment: Extract phosphorylated peptides using methods like TiO₂, Fe-NTA
6. LC-MS/MS Analysis: Use high-resolution mass spectrometry platforms (e.g., Orbitrap Fusion Lumos)
7. Data Analysis: Perform quantitative and statistical analysis using software like MaxQuant and Perseus
V. Solutions Provided by Biotech Co., Ltd.
Biotech Co., Ltd. providescomprehensive solutions for SILAC-based phosphoproteomics, including but not limited to:
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customization of high-purity heavy-labeled amino acids and optimization of cell labeling
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phosphopeptide enrichment and multi-round enrichment processes
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Orbitrap high-resolution mass spectrometry platform analysis
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quantitative data mining services based on MaxQuant/Perseus
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phosphorylation site annotation, pathway enrichment, and network construction
The service projects cover cancer signal research, kinase inhibitor target screening, receptor pathway kinetic modeling, etc., assisting research customers inprotein modification regulatory mechanism researchto achieve high-level results.
SILAC technology, with itsendogenous labeling, precise quantification, and compatibility with various modification enrichment strategiesadvantages, has become one of the core methods in phosphorylation proteomics research. In the future, as mass spectrometry technology and data analysis tools continue to advance, SILAC is expected to play a key role in broader PTM research. If you plan to conduct SILAC quantitative phosphorylation research, feel free to contact Biotech Co., Ltd. We will assist your phosphorylation modification research with high-quality technical services and project management.
