SILAC Quantitative Proteomics Comprehensive Analysis: From Isotope Labeling to Mass Spectrometry Quantification

Among various quantitative proteomics techniques, SILAC (Stable Isotope Labeling by Amino acids in Cell culture) stands out for its high precision and low bias, playing a significant role in comparing protein expression at the cellular level. By introducing stable isotope-labeled amino acids during cell culture, SILAC achieves "natural" labeling of proteins, avoiding systematic errors caused by chemical modifications outside the body. This article systematically analyzes the complete process of SILAC from cell labeling, sample preparation, mass spectrometry analysis to data processing, helping researchers fully master the key points of this technology.

 

I. Principle of SILAC Quantitative Proteomics

SILAC utilizes stable isotope-labeled amino acids such as 13C or 15N (e.g., 13C6-lysine, 13C6-arginine) to feed cells in a medium without endogenous amino acids, allowing the incorporation of heavy labeled amino acids into the peptide chain during protein synthesis. In experiments, control group cells are usually cultured in light labeled medium, while treatment group cells are cultured under heavy labeled conditions. After collection, they are mixed in equal amounts and uniformly subjected to enzymatic digestion and LC-MS/MS analysis. Mass spectrometry detects protein expression quantitatively through the relative peak area ratio of light/heavy peptides.

 

II. Full Process of SILAC Quantitative Proteomics

1. Cell Line Selection

The premise of SILAC is that cells can grow and divide normally in serum-free, amino acid-free basal medium (such as DMEM-SILAC). Common cell lines such as HeLa, HEK293, and A549 have been extensively validated in SILAC experiments, demonstrating good labeling efficiency and stable protein expression.

 

2. Medium Configuration and Replacement

Light medium contains natural lysine and arginine; heavy medium replaces them with 13C6-lysine and 13C6,15N4-arginine. Labeling usually requires continuous passage for more than 6 cycles (about 5-7 days) to achieve >97% isotope incorporation rate.

 

3. Verification of Labeling Efficiency

Preliminary LC-MS/MS analysis or using MALDI mass spectrometry can evaluate isotope incorporation efficiency. If it is below 95%, it is recommended to extend the culture time or optimize cell conditions to ensure the reliability of subsequent data.

 

4. Sample Preparation and Protein Extraction

After light and heavy labeling are completed, mix cell lysates in proportion according to total protein amount. Lysis usually uses RIPA buffer or SDS lysis buffer, with protease inhibitors added to prevent degradation. The extracted protein samples need to be quantitatively consistent, and then subjected to reduction, alkylation, and trypsin digestion treatment.

 

5. LC-MS/MS Mass Spectrometry Analysis

Digest products are desalted using C18 solid phase extraction columns and injected into high-resolution nano LC-MS/MS systems (such as Orbitrap Fusion, Q Exactive, etc.). Mass spectrometry parameters need to be optimized to resolve small mass differences between light/heavy peptides. DDA (Data Dependent Acquisition) mode is generally used to obtain MS1 and MS2 data, achieving protein identification and quantification.

 

6. Data Processing and Quantitative Analysis

MaxQuant is the mainstream software for SILAC data analysis, supporting automatic identification and paired quantification of light/heavy peptides. It can output relative changes in protein expression, significance analysis, and differential protein lists. Further statistical analysis, functional enrichment, and pathway annotation can be performed using Perseus.

 

III. Advantages and Limitations of SILAC Quantitative Proteomics

1. Advantages

(1) Endogenous labeling, avoiding errors from exogenous modifications

(2) Good quantitative reproducibility, suitable for time series and intervention experiments

(3) Few missing values, strong quantitative consistency

 

2. Limitations

(1) Only applicable to cultivable cell lines

(2) Not suitable for complex samples like tissues and blood

(3) High cost of labeled amino acids, long experimental cycle

 

Biotech Pack Technology has established an efficient and stable SILAC quantitative proteomics platform, covering the entire process from cell labeling, sample preparation to high-resolution mass spectrometry analysis and data interpretation. We have optimized SILAC culture systems for various commonly used cell lines and, combined with the Orbitrap mass spectrometry platform and MaxQuant analysis process, provide precise and reliable data support for projects like drug screening and signal pathway research.

 

As a metabolic labeling protein quantification strategy, SILAC has played an important role in cell mechanism research due to its high precision and endogenous consistency. In the future, SILAC quantitative proteomics is expected to combine with phosphoproteomics, interactomics, and other omics methods to expand more diverse application scenarios. Introducing a mature SILAC platform, such as the customized services of Biotech Pack Technology, at the initial stage of project planning will greatly enhance data quality and experimental efficiency.

 

Biotech Pack Technology—A high-quality service provider of bioproduct characterization and multi-omics biological mass spectrometry testing

 

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