Stable Isotope Labeling by Amino acids in Cell culture

Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is a protein quantification technique based on the principle of metabolic isotope endogenous integration. This method involves adding amino acids labeled with stable isotopes, such as [^13C_6]-lysine or [^13C_6^15N_4]-arginine, to the cell culture medium. As cells grow, they naturally incorporate these labeled amino acids into synthesized proteins, resulting in distinguishable 'light' and 'heavy' peptide segments in mass spectrometry analysis. This endogenous labeling method requires no chemical modifications and minimizes inter-sample errors, enabling high-precision relative quantification. SILAC is a classic strategy in differential proteomic analysis at the cellular level. Compared to other protein quantification strategies, SILAC offers significant technical advantages: it provides high quantification precision and avoids reaction efficiency differences that may arise during chemical labeling. The complete isotopic substitution before sample mixing ensures consistent sample treatment, greatly reducing systematic errors. Additionally, since labeling occurs at the metabolic level, SILAC can be used to track protein synthesis and degradation rates, supporting dynamic proteomics analysis. These characteristics make it an ideal technical platform for phenotypic association studies, drug mechanism research, and intervention effect evaluation in cell models.

 

The experimental design of Stable Isotope Labeling by Amino acids in Cell culture typically involves two or more groups of cells: one group grown in a 'light' medium containing natural amino acids, and another group in a 'heavy' medium containing the corresponding stable isotope-labeled amino acids. The cell culture time should cover a sufficient number of doubling cycles (usually 5-7 generations) to ensure that most cellular proteins are composed of labeled amino acids. After labeling is complete, cells from different treatment groups are lysed, equally mixed, and subjected to protein extraction, digestion, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In the mass spectrum, labeled peptides show a specific mass shift relative to unlabeled peptides, allowing accurate quantification through relative abundance calculation. The greatest advantage of SILAC is that the labeling occurs during the physiological process of the cell, without affecting protein structure and function, thus preserving biological authenticity to the greatest extent.

 

Although SILAC has many advantages, it also has certain limitations. For example, it is mainly applicable to mammalian cell lines that can stably grow in culture medium for extended periods and is not suitable for primary cells or tissue samples. Additionally, labeling efficiency is related to cellular metabolic activity, requiring strict control of the labeling period and amino acid concentration in experimental design. For low-abundance proteins that are not fully labeled, mass spectrometry analysis still relies on high-sensitivity equipment and optimized database search strategies to ensure data reliability.

 

Biotech Pack relies on a professional proteomics platform and extensive mass spectrometry analysis experience to provide high-standard SILAC/Dimethyl-based quantitative proteomics analysis services for research clients, including experimental design, sample processing, data acquisition, and in-depth interpretation.

 

Biotech Pack - Characterization of Biological Products, a quality service provider for multi-omics mass spectrometry detection.

 

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Quantitative Proteomics Analysis based on SILAC/Dimethyl Labeling