Quantitative proteomics based on mass spectrometry has become a powerful tool in biological research. However, mass spectrometry is inherently unsuitable for protein quantification analysis because different proteins and peptides have different ionization efficiencies. Stable isotope labeling of amino acids (such as 2H(D), 13C, 15N, and 18O) introduced into proteins can effectively address this issue.
Stable isotope labeling by amino acids in cell culture (SILAC) is a simple, stable, and powerful method in quantitative proteomics based on mass spectrometry (MS). It was first used in quantitative proteomics by Ong et al. from the Mann laboratory in Denmark in 2022. SILAC provides accurate relative quantification without the need for any chemical derivatization or other manipulations and is widely used in research fields such as cell biology, biochemistry, and pharmacology.
Basic Principles and Processes of SILAC
In the realm of quantitative proteomics, the technique of stable isotope labeling of amino acids in cell culture (SILAC) stands out for its elegant simplicity and profound implications. This method relies on the metabolic incorporation of stable isotope-labeled amino acids into proteins across entire cellular proteomes. Two sets of cells are cultured simultaneously: one set in a medium containing normal, or "light," amino acids, while the other set is cultured in a medium enriched with stable isotope-labeled, or "heavy," amino acids.
Over several generations of cell passage, typically spanning 5-6 rounds, these stable isotope-labeled amino acids gradually replace their natural counterparts within the newly synthesized proteins. This metabolic incorporation process occurs seamlessly, with the labeled amino acids becoming integral components of the growing polypeptide chains. As a result, the only discernible distinction between the proteins derived from the "light" and "heavy" cell populations is a shift in molecular weight, without any alterations in chemical properties or functionalities.
Once the stable isotope-labeled amino acids have been fully integrated into the cellular proteomes, the two sets of cells are often mixed together. This merging of cellular populations serves to create a complex sample containing a blend of proteins labeled with "light" and "heavy" isotopes, representing distinct experimental conditions or biological states. Subsequently, the proteins are extracted from the mixed cell population and enzymatically digested into peptides, which are then subjected to analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
LC-MS/MS analysis enables the high-throughput detection and characterization of peptides based on their mass-to-charge ratios and fragment ion spectra. By sequentially separating peptides within a complex mixture using liquid chromatography and subsequently subjecting them to tandem mass spectrometry for fragmentation and identification, LC-MS/MS provides a comprehensive snapshot of the proteomic landscape within the sample. Importantly, the incorporation of stable isotope labeling allows for the precise quantification of protein abundance levels between the "light" and "heavy" conditions, facilitating comparative proteomic analyses and the identification of differentially expressed proteins.
Overview of the SILAC experiment (Ong et al., 2002)
The SILAC experiment comprises two distinct phases — the adaptation (a) and experimental (b) phases.
(a) During the adaptation phase, cells are grown in both light and heavy SILAC media until the heavy cells are fully incorporated with heavy amino acids (depicted by red stars). This allows for complete differentiation between the two SILAC cell pools via MS (black dots and red stars representing light and heavy SILAC peptides, respectively), which can then be mixed and treated as a single sample. The adaptation phase may involve cell proliferation to reach the required number of dishes for the experiment.
(b) In the second phase, the two cell populations are subjected to different treatments to induce changes in the proteome. After mixing the samples, sub-proteomes can be enriched through enrichment steps or other purification methods, digested into peptides as a single pool, and subjected to protein identification and quantitative analysis via MS.
Selection of Isotope-Labeled Amino Acids in SILAC Technology
Ideally, SILAC amino acids should be essential for cell survival, ensuring that the only source of specific amino acids is the culture medium. Leucine, lysine, and arginine are essential amino acids that have been used in SILAC.
In early SILAC studies, deuterated (2H) leucine was chosen as the labeled amino acid. However, the chromatographic shift of deuterated peptides during reverse-phase chromatography affected the accuracy of quantification. Subsequently, amino acids labeled with 13C or 15N were used because these SILAC peptides undergo co-elution during LC-MS/MS analysis.
Although arginine (R) is not an essential amino acid, it has been shown to be essential for many cultured cell lines and has been successfully used for SILAC labeling. Since proteins need to be "cut" into peptide segments by trypsin before subsequent protein mass spectrometry analysis, and trypsin specifically "cuts" at lysine (K) and arginine (R) residues, resulting in over 95% of peptides ending with K and R residues, which enriches the mass spectrometry quantitative information and ensures more precise quantification results. Therefore, an increasing number of researchers are using combinations of 13C and 15N-labeled arginine and lysine as labeled amino acids.
Tyrosine is another non-essential amino acid used in SILAC. Heavy-labeled tyrosine is employed to identify substrates of tyrosine kinases, studying the dynamic changes in protein tyrosine phosphorylation.
Quantitative Proteomics Based on SILAC
Quantitative proteomics based on SILAC can be used to identify specific interacting proteins in exogenous protein-protein interactions (PPIs), endogenous PPIs, or specific interaction proteins in inducible PPIs.
Quantitative interaction proteomics with SILAC (Chen et al., 2015)
When studying exogenous protein complexes, wild-type cells or cells expressing affinity-tagged proteins are grown in either light or heavy media. Immunoprecipitation of protein complexes is then performed from mixtures of lysates obtained from light and heavy cells.
For the investigation of endogenous protein complexes, cells grown in either light or heavy media are subjected to RNAi knockdown of the target protein. Subsequently, immunoprecipitation of protein complexes is carried out using corresponding antibodies from mixtures of lysates obtained from light and heavy cells.
In the case of inducible PPIs, protein complexes are induced by specific stimuli in cells grown in either light or heavy media. Immunoprecipitation purification is then performed from mixtures of lysates obtained from light and heavy cells. Following the acquisition of protein complexes, proteins are digested into peptides and analyzed using LC-MS/MS. Specific interacting proteins or nonspecific background proteins can be distinguished based on their SILAC ratios.
Advantages of SILAC Technology
Efficiency: SILAC utilizes in vivo labeling, where cells are cultured in media containing isotopically labeled amino acids. This approach ensures high labeling efficiency, often reaching close to 100%. Every protein synthesized by the cells incorporates the labeled amino acids, resulting in complete and uniform labeling throughout the proteome.
Precision in Quantification: SILAC provides a wide linear range of quantification, enabling accurate measurement of protein abundance across a dynamic range of concentrations. This reduces errors associated with sample preparation, experimental procedures, and instrument variations. Consequently, SILAC offers excellent quantitative repeatability, allowing for reliable comparisons between different experimental conditions or biological samples.
High Throughput: SILAC technology is highly scalable, allowing researchers to simultaneously analyze and quantify hundreds to thousands of proteins in a single experiment. This high throughput capability is particularly advantageous for large-scale proteomics studies aiming to comprehensively characterize protein expression patterns or identify changes in protein abundance under various conditions.
High Sensitivity: SILAC requires relatively small amounts of sample input, typically only a few tens of micrograms of protein per sample. Despite the low sample requirements, SILAC achieves high sensitivity in protein detection and quantification. This sensitivity is particularly valuable when analyzing limited or precious biological samples, such as clinical specimens or samples obtained from rare cell populations.
In Vivo Labeling: SILAC employs labeling within living cells, which closely mimics physiological conditions. By incorporating isotopically labeled amino acids during protein synthesis within the cellular environment, SILAC preserves the native state of proteins and their post-translational modifications. This in vivo labeling approach ensures that the experimental results reflect the true biological context, enhancing the relevance and reliability of the findings.
Compatibility: SILAC technology is compatible with a wide range of cell types and organisms that can be cultured in standard cell culture media such as DMEM (Dulbecco's Modified Eagle Medium) or RPMI 1640. This flexibility allows researchers to apply SILAC labeling to diverse biological systems, including mammalian cells, bacteria, and even certain model organisms, expanding the scope of SILAC applications in proteomics research.
References
- Ong, Shao-En, et al. "Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics." Molecular & cellular proteomics 1.5 (2002): 376-386.
- Chen, Xiulan, et al. "Quantitative proteomics using SILAC: Principles, applications, and developments." Proteomics 15.18 (2015): 3175-3192.
- Krijgsveld, Jeroen, et al. "Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics." Nature biotechnology 21.8 (2003): 927-931.
