SILAC Phosphoproteomics
SILAC phosphoproteomics represents an advanced methodology that integrates stable isotope labeling with phosphoprotein analysis to investigate the dynamic alterations of phosphorylated proteins within cells. Phosphorylation is a prevalent post-translational modification, playing critical roles in cellular signaling, metabolic regulation, and cell cycle control. Investigating changes in phosphorylated proteins is crucial for elucidating cellular signaling pathways, disease mechanisms, and drug effects. SILAC phosphoproteomics not only allows precise quantitation of phosphorylation site alterations but also aids in identifying pivotal nodes and pathways within regulatory networks, offering robust support for both fundamental research and clinical applications. This approach facilitates a comprehensive evaluation of phosphorylated proteins in cells and holds promise across cancer research, neuroscience, and immunology. For instance, in cancer studies, anomalous phosphorylation patterns are closely linked to tumorigenesis and metastasis. SILAC phosphoproteomics assists researchers in pinpointing potential cancer-related biomarkers and therapeutic targets. In neuroscience, phosphorylation is integral to neuronal signal transduction and synaptic plasticity, enhancing our understanding of neurological disorders like Alzheimer's and Parkinson's diseases.
Technical Process
The SILAC phosphoproteomics workflow initiates with cell culture, where stable isotope-labeled amino acids are incorporated into cellular proteins via the culture medium. Following specific cellular treatments, such as drug exposure or environmental modifications, researchers can observe changes in protein phosphorylation. Post-cell lysis, proteins are extracted and enzymatically cleaved into peptides. Phosphopeptides are then selectively enriched to boost the sensitivity and accuracy of subsequent mass spectrometry analyses. During mass spectrometry, SILAC-labeled and control samples are co-analyzed, enabling precise quantitation of phosphorylation changes through mass-to-charge ratio (m/z) comparison. The final stage involves bioinformatics analyses to interpret mass spectrometry data, identifying differentially expressed phosphorylation sites and their biological implications.
Advantages and Challenges
SILAC phosphoproteomics offers significant advantages in sensitivity and quantitative precision. Unlike traditional methods, SILAC facilitates the comparative analysis of multiple samples within a single experiment, minimizing experimental error and enhancing result reliability. Additionally, it circumvents issues associated with chemical labeling or antibody specificity. Nevertheless, challenges persist, such as the requirement for prolonged cell culture in labeled amino acids, which may not be feasible for all cell types. Moreover, while SILAC is predominantly used for in vitro studies, its application to complex in vivo tissues requires further refinement. Effective enrichment and identification of phosphopeptides remain critical to experimental success, necessitating meticulous experimental design and optimization.
MtoZ Biolabs is committed to delivering high-quality label-based protein quantification services, including iTRAQ, TMT, and SILAC. Our team, equipped with extensive expertise and cutting-edge technology, provides tailored solutions to meet diverse research needs. We prioritize both data accuracy and the biological relevance of findings, ensuring maximum research value for our clients. Whether in basic research or applied development, MtoZ Biolabs stands ready to collaborate in advancing the frontiers of proteomics.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
