SILAC Quantitative Proteomics Principles
Stable Isotope Labeling with Amino acids in Cell culture (SILAC) combined with liquid chromatography-tandem mass spectrometry is a quantitative proteomics technique that uses isotopic labeling to allow high-precision quantitative analysis of proteins via mass spectrometry. It has become a critical tool in modern biomedical research. Below is a detailed explanation of the principles of SILAC quantitative proteomics.
1. Basic Principles of SILAC Quantitative Proteomics
The basic principle of SILAC technology involves adding amino acids containing stable isotopes to the cell culture medium. These isotopically-labeled amino acids are incorporated endogenously into proteins during cell division and growth. A mass spectrometer is then used to perform quantitative analysis of both labeled and unlabeled proteins.
2. SILAC Sample Preparation
Key steps in SILAC sample preparation include cell labeling, protein extraction and digestion, and liquid chromatography separation. Cell labeling is a critical step in the process, requiring the addition of amino acids with stable isotopes to ensure protein isotopic labeling.
3. Enhancing Sensitivity of SILAC Quantification
Since the content of protein labeled with stable isotopes is relatively low, enhancing the sensitivity of the mass spectrometer is crucial. This can be achieved by optimizing liquid chromatography separation and mass spectrometry acquisition parameters, along with using efficient ion sources and precise mass analyzers.
4. Reducing Complexity in SILAC Quantification
Samples may contain a large number of proteins, so it's necessary to reduce sample complexity. This can be done by employing efficient protein separation and concentration techniques, and selecting appropriate enzymes for protein digestion.
5. Utilizing Multidimensional Mass Spectrometry Techniques
Multidimensional mass spectrometry techniques can further enhance the accuracy and reliability of SILAC quantification. For example, using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) and data-independent acquisition (DIA) allows for the simultaneous acquisition of more protein information during mass spectrometry analysis.
6. Integration of Data Analysis and Bioinformatics
Mass spectrometry data generated by SILAC quantification needs to be analyzed and interpreted using bioinformatics tools. This includes normalization of protein quantification data, localization and matching of isotopic peaks, and protein localization and function prediction.
BiotechPack, A Biopharmaceutical Characterization and Multi-Omics Mass Spectrometry (MS) Services Provider
Related Services:
Quantitative Proteome Analysis Based on SILAC/Dimethyl Labeling
Label-Based Protein Quantification Techniques - iTRAQ, TMT, SILAC
How to order?
